U.S. patent number 7,582,463 [Application Number 11/564,476] was granted by the patent office on 2009-09-01 for non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes.
This patent grant is currently assigned to Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo. Invention is credited to Shigeharu Fukuda, Michio Kubota, Kazuhiko Maruta, Toshio Miyake, Takuo Yamamoto.
United States Patent |
7,582,463 |
Yamamoto , et al. |
September 1, 2009 |
Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme,
and process for producing saccharides using the enzymes
Abstract
A non-reducing saccharide-forming enzyme and a
trehalose-releasing enzyme, which have an optimum temperature in a
medium temperature range, i.e., a temperature of over 40 or
45.degree. C. but below 60.degree. C.; and an optimum pH in an acid
pH range, i.e., a pH of less than 7. The two-types of enzymes can
be obtained in a desired amount, for example, by culturing in a
nutrient culture medium microorganisms capable of producing the
enzymes or by recombinant DNA technology.
Inventors: |
Yamamoto; Takuo (Okayama,
JP), Maruta; Kazuhiko (Okayama, JP),
Kubota; Michio (Okayama, JP), Fukuda; Shigeharu
(Okayama, JP), Miyake; Toshio (Okayama,
JP) |
Assignee: |
Kabushiki Kaisha Hayashibara
Seibutsu Kagaku Kenkyujo (Okayama, JP)
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Family
ID: |
27281623 |
Appl.
No.: |
11/564,476 |
Filed: |
November 29, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070281346 A1 |
Dec 6, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09435770 |
Nov 8, 1999 |
7186535 |
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09392253 |
Sep 9, 1999 |
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Foreign Application Priority Data
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Sep 11, 1998 [JP] |
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1998-258394 |
Dec 11, 1998 [JP] |
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1998-352252 |
Jan 26, 1999 [JP] |
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1999-016931 |
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Current U.S.
Class: |
435/201;
536/23.2; 435/320.1; 435/252.3; 435/200; 435/183 |
Current CPC
Class: |
C12N
9/2402 (20130101); C12P 19/18 (20130101); C12P
19/04 (20130101); C12Y 302/01001 (20130101); C12Y
302/01003 (20130101); C12Y 302/01002 (20130101); C12P
19/14 (20130101); C12Y 302/0102 (20130101); C12N
9/90 (20130101) |
Current International
Class: |
C12N
9/00 (20060101); C07H 21/04 (20060101); C12N
1/20 (20060101); C12N 15/00 (20060101); C12N
9/24 (20060101); C12N 9/26 (20060101) |
Field of
Search: |
;435/183,193,200,252.3,320.1 ;536/23.2 |
References Cited
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Primary Examiner: Fronda; Christian L
Attorney, Agent or Firm: Browdy and Neimark, PLLC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of copending parent
application Ser. No. 09/392,253, filed Sep. 9, 1999, the entire
contents of which being hereby incorporated herein by reference.
Claims
What is claimed is:
1. A purified trehalose-releasing enzyme which hydrolyzes a
non-reducing saccharide having a trehalose structure as an end unit
to release said trehalose end unit from the rest of said
non-reducing saccharide and which has an optimum temperature of
over 45.degree. C. but below 60.degree. C., wherein said enzyme
comprises the amino acid sequence of SEQ ID NO:9.
2. The purified trehalose-releasing enzyme of claim 1, wherein said
enzyme consists of the amino acid sequence of SEQ ID NO:9.
3. The purified trehalose-releasing enzyme of claim 1, which has
the following physicochemical properties: (1) Action Hydrolyzing a
non-reducing saccharide having a trehalose structure as an end unit
to release said trehalose end unit from the rest of said
non-reducing saccharide; (2) Molecular weight About 62,000.+-.5,000
daltons on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE); (3) Optimum temperature About
50.degree. C. to about 55.degree. C. when incubated at pH 6.0 for
30 min; (4) Optimum pH About 6.0 when incubated at 50.degree. C.
for 30 min; (5) Thermal stability Stable up to a temperature of
about 50.degree. C. when incubated at pH 7.0 for 60 min; and (6) pH
stability Stable at pHs of about 4.5 to about 10.0 when incubated
at 4.degree. C. for 24 hours.
4. The purified trehalose-releasing enzyme of claim 1, which is
isolated from a microorganism.
5. The purified trehalose-releasing enzyme of claim 4, wherein said
microorganism is a member of the genus Arthrobacter.
6. The purified trehalose-releasing enzyme of claim 4, wherein said
microorganism is Arthrobacter sp. S34, deposited under accession
no. FERM BP-6450.
7. The purified trehalose-releasing enzyme of claim 1 obtainable
from Arthrobacter sp. S34, deposited under accession no. FERM
BP-6450.
8. A purified trehalose-releasing enzyme which hydrolyzes a
non-reducing saccharide having a trehalose structure as an end unit
to release said trehalose end unit from the rest of said
non-reducing saccharide and which has an optimum temperature of
over 45.degree. C. but below 60.degree. C., wherein said enzyme is
a fragment of an enzyme consisting of the amino acid sequence of
SEQ ID NO:9.
9. The purified trehalose-releasing enzyme of claim 8, wherein said
fragment comprises the amino acid sequence of SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12 or SEQ ID NO:13.
10. The purified trehalose-releasing enzyme of claim 8, wherein
said fragment comprises the amino acid sequence of SEQ ID NO:14,
SEQ ID NO:15, or SEQ ID NO:16.
11. The purified trehalose-releasing enzyme of claim 8, which has
the following physicochemical properties: (1) Action Hydrolyzing a
non-reducing saccharide having a trehalose structure as an end unit
to release said trehalose end unit from the rest of said
non-reducing saccharide; (2) Molecular weight About 62,000.+-.5,000
daltons on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE); (3) Optimum temperature About
50.degree. C. to about 55.degree. C. when incubated at pH 6.0 for
30 min; (4) Optimum pH About 6.0 when incubated at 50.degree. C.
for 30 min; (5) Thermal stability Stable up to a temperature of
about 50.degree. C. when incubated at pH 7.0 for 60 min; and (6) pH
stability Stable at pHs of about 4.5 to about 10.0 when incubated
at 4.degree. C. for 24 hours.
12. The purified trehalose-releasing enzyme of claim 8, which is
isolated from a microorganism.
13. The purified trehalose-releasing enzyme of claim 8 obtainable
from Arthrobacter sp. S34, deposited under accession no. FERM
BP-6450.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a non-reducing saccharide-forming
enzyme, a trehalose-releasing enzyme, and a process for producing
saccharides using the enzymes.
2. Description of the Prior Art
Trehalose is a disaccharide consisting of two moles of glucose
bound at their reducing residues, and is widely found in nature,
for example in microorganisms, fungi, algae, insects, Crustacea,
etc. Since the saccharide has long been known as a useful
saccharide substantially free of reducibility and having a
satisfactory moisture-retaining action, it has been expected to use
in extensive fields including foods, cosmetics, and
pharmaceuticals. However, no efficient production of the saccharide
was established, and this narrows the use of trehalose in spite of
its outstanding expectation. Thus supply of trehalose in a lower
cost is greatly expected.
As a proposal for such an expectation, the present inventors had
already established a process for enzymatically producing trehalose
from material starches through their energetic studies. The process
is characterized by a step of subjecting reducing partial starch
hydrolysates to the action of a non-reducing saccharide-forming
enzyme, which forms a non-reducing saccharide having a trehalose
structure as an end unit from reducing partial starch hydrolysates,
and to the action of a trehalose-releasing enzyme which acts on a
non-reducing saccharide having a trehalose structure as an end unit
in order to hydrolyze and release trehalose from the rest of the
non-reducing saccharide. These enzymes and processes thereof are
disclosed in Japanese Patent Kokai Nos. 143,876/95, 213,283/95,
322,883/95, 298,880/95, 66,187/96, 66,188/96, 73,504/96, 84,586/96,
and 336,388/96, applied for by the same applicant as the present
invention. Thus, a low-cost production of trehalose was
attained.
During the studies, they found an original finding that the
non-reducing saccharide-forming enzyme can be applied for a novel
production of non-reducing saccharides that can overcome
conventional drawback residing in reducing partial starch
hydrolysates. As a problem, reducing partial starch hydrolysates
such as dextrins and maltooligosaccharides have advantageous
features that they can be used as sweeteners and
energy-supplementing saccharide sources, but as a demerit they are
highly reactive with substances because of their reducibility and
are susceptible to browning reaction when coexisted with amino
acids and/or proteins and to readily deteriorate their quality. To
overcome such a problem, it is only known a method to convert
reducing partial starch hydrolysates into sugar alcohols using a
high-pressure hydrogenation method, etc. In actual use, the method,
however, needs much heats and instruments constructed under
consideration of safety in view of the use of hydrogen, resulting
in a higher cost and much labor cost. On the contrary, the
aforesaid non-reducing saccharide-forming enzyme as mentioned
previously acts on reducing partial starch hydrolysates and forms
non-reducing saccharide having a trehalose structure as an end
unit, and the reaction proceeds under a relatively-mild condition
due to its enzymatic reaction. Using the action of the enzyme, the
present inventors established a novel efficient process for
non-reducing saccharides using the enzyme, that can overcome
conventional drawback residing in reducing partial starch
hydrolysates. Because of these findings, the development of
applicable uses for trehalose and non-reducing saccharides have
become to be flourished in various fields, and this diversifies the
uses of these saccharides and now remarkably increases the demands
of the saccharides in a wide variety of fields.
Under these circumstances, a more efficient process for producing
trehalose and non-reducing saccharides having a trehalose structure
has been more expected in this art. A key to such an expectation is
to establish a non-reducing saccharide-forming enzyme and a
trehalose-releasing enzyme with various optimum conditions, and to
provide a wide variety of sources for such enzymes usable in the
production of the saccharides. Thus, an optimum enzyme can be
chosen from various types of enzymes depending on the optimum
conditions of another enzymes usable in combination with the above
enzymes to produce the desired saccharides, as well as on
installations and final uses of the saccharides produced, resulting
in an efficient production of the saccharides. Conventionally known
non-reducing saccharide-forming enzymes can be grouped into those
having optimum temperatures of relatively-lower temperatures of
about 40.degree. C. or lower, and those having optimum temperatures
of relatively-higher temperatures of about 60.degree. C. or higher.
While conventionally known trehalose-releasing enzymes can be
grouped into those having optimum temperatures in a
relatively-lower temperature range, about 45.degree. C. or lower,
and those having optimum temperatures in a relatively-higher
temperature range, about 60.degree. C. or higher. However, any
non-reducing saccharide-forming enzyme and a trehalose-releasing
enzyme having an optimum temperature in a medium temperature range,
about 50.degree. C., have never yet been opened.
Among saccharide-related enzymes used in the production of
saccharides from starch materials, enzymes as a major group have an
optimum temperature in a medium temperature range. Such enzymes may
be required in the process for producing the aforesaid trehalose
and non-reducing saccharides; No non-reducing saccharide-forming
enzyme and no trehalose-releasing enzyme, which have an optimum
temperature in a medium temperature range, have not yet been
established so that there has not yet been realized a process for
producing saccharides in a sufficient yield using either or both of
these enzymes together with the above saccharide-related enzymes.
Depending on installations for producing saccharides and final uses
of them, there have been required enzymes having an optimum
temperature in a medium temperature range in their enzymatic
reactions. It is far from saying that it has established a process
for producing saccharides in a satisfactorily-high yield using a
non-reducing saccharide-forming enzyme and a trehalose-releasing
enzyme. As described above the establishment of a non-reducing
saccharide-forming enzyme and a trehalose-releasing enzyme having
an optimum temperature in a medium temperature range, and a process
for producing saccharides comprising non-reducing saccharides are
in great demand.
SUMMARY OF THE INVENTION
In view of this, the first object of the present invention is to
provide a non-reducing saccharide-forming enzyme having an optimum
temperature in a medium temperature range.
The second object of the present invention is to provide a DNA
encoding the non-reducing saccharide-forming enzyme.
The third object of the present invention is to provide a process
for producing the non-reducing saccharide-forming enzyme.
The fourth object of the present invention is to provide a
trehalose-releasing enzyme having an optimum temperature in a
medium temperature range.
The fifth object of the present invention is to provide a DNA
encoding the trehalose-releasing enzyme.
The sixth object of the present invention is to provide a process
for producing the trehalose-releasing enzyme.
The seventh object of the present invention is to provide a
microorganism capable of producing the non-reducing
saccharide-forming enzyme and/or the trehalose-releasing
enzyme.
The eighth object of the present invention is to provide a process
for producing saccharides comprising non-reducing saccharides,
which uses the non-reducing saccharide-forming enzyme and/or the
trehalose-releasing enzyme.
In order to attain the above objects, the present inventors
extensively screened microorganisms, that can overcome the objects,
in soils. As a result, they found that a microorganism newly
isolated from a soil in Ako-shi, Hyogo, Japan, produced enzymes
that can solve the above objects. The present inventors isolated
separatory the desired non-reducing saccharide-forming enzyme and
trehalose-releasing enzyme from the microorganism, and then
identified their properties, revealing that the enzymes both had an
optimum temperature in a medium temperature range. The
identification of the microorganism confirmed that it was a novel
microorganism of the genus Arthrobacter, and named Arthrobacter sp.
S34. The microorganism was deposited on Aug. 6, 1998, in the
National Institute of Bioscience and Human-Technology Agency of
Industrial Science and Technology, Higashi 1-1-3, Tsukuba-shi,
Ibaraki, Japan, and accepted and has been maintained by the
institute under the accession number of FERM BP-6450.
The present inventors continued studying, isolated DNAs encoding
the above-identified enzymes from the microorganism, Arthrobacter
sp. S34, FERM BP-6450, decoded the nucleotide sequences, and
determined the amino acid sequences of the enzymes. The inventors
confirmed that Arthrobacter sp. S34, FERM BP-6450, and
transformants, into which the DNAs obtained in the above had been
introduced in a usual manner, produced desired amounts of enzymes.
It was also confirmed that the enzymes thus obtained can be
advantageously used in producing saccharides which comprise
trehalose and non-reducing saccharides having a trehalose structure
in a medium temperature range. The present invention was made based
on these findings.
The first object of the present invention is solved by a novel
non-reducing saccharide-forming enzyme that forms a non-reducing
saccharide having a trehalose structure as an end unit from
reducing partial starch hydrolysates, and has an optimum
temperature in a medium temperature range.
The second object of the present invention is solved by a DNA
encoding the non-reducing saccharide-forming enzyme.
The third object of the present invention is solved by a process
for producing the non-reducing saccharide-forming enzyme,
characterized in that it comprises the steps of culturing a
microorganism capable of producing the enzyme, and collecting the
produced enzyme from the culture.
The fourth object of the present invention is solved by a novel
trehalose-releasing enzyme which specifically hydrolyses a
non-reducing saccharide having a trehalose structure as an end unit
and a glucose polymerization degree of at least 3 to release
trehalose from the rest of the non-reducing saccharide, and which
has an optimum temperature in a medium temperature range.
The fifth object of the present invention is solved by a DNA
encoding the trehalose-releasing enzyme.
The sixth object of the present invention is solved by a process
for producing the trehalose-releasing enzyme, characterized in that
it comprises the steps of culturing a microorganism capable of
producing the enzyme, and collecting the produced enzyme from the
culture.
The seventh object of the present invention is solved by a
microorganism selected from Arthrobacter sp. S34, FERM BP-6450, and
mutants thereof.
The eighth object of the present invention is solved by a process
for producing saccharides, comprising the steps of allowing the
either or both of the above enzymes to act on reducing partial
starch hydrolysates to produce non-reducing saccharides, and
collecting the non-reducing saccharides or saccharide compositions
having a relatively-low reducibility and containing the
non-reducing saccharides.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1 is a figure that shows the influence of temperature on the
activity of a non-reducing saccharide-forming enzyme from
Arthrobacter sp. S34, FERM BP-6450, according to the present
invention.
FIG. 2 is a figure that shows the influence of pH on the activity
of a non-reducing saccharide-forming enzyme from Arthrobacter sp.
S34, FERM BP-6450, according to the present invention.
FIG. 3 is a figure that shows the influence of temperature on the
stability of a non-reducing saccharide-forming enzyme from
Arthrobacter sp. S34, FERM BP-6450, according to the present
invention.
FIG. 4 is a figure that shows the influence of pH on the stability
of a non-reducing saccharide-forming enzyme from Arthrobacter sp.
S34, FERM BP-6450, according to the present invention.
FIG. 5 is a restriction map of the recombinant DNA pGY1 according
to the present invention. The bold line shows the nucleotide
sequence from Arthrobacter sp. S34, FERM BP-6450. The black arrow
within the bold line shows a nucleotide sequence encoding the
present non-reducing saccharide-forming enzyme, while the oblique
arrow shows a nucleotide sequence encoding the present
trehalose-releasing enzyme.
FIG. 6 is a restriction map of the recombinant DNA pGY2 according
to the present invention. The bold line shows the nucleotide
sequence from Arthrobacter sp. S34, FERM BP-6450. The black arrow
within the bold line shows a nucleotide sequence encoding the
present non-reducing saccharide-forming enzyme.
FIG. 7 is a restriction map of the recombinant DNA pGY3 according
to the present invention. The black arrow shows the nucleotide
sequence, encoding the present non-reducing saccharide-forming
enzyme, from Arthrobacter sp. S34, FERM BP-6450.
FIG. 8 is a figure that shows the influence of temperature on the
activity of a trehalose-releasing enzyme from Arthrobacter sp. S34,
FERM BP-6450, according to the present invention.
FIG. 9 is a figure that shows the influence of pH on the activity
of a trehalose-releasing enzyme from Arthrobacter sp. S34, FERM
BP-6450, according to the present invention.
FIG. 10 is a figure that -shows the influence of temperature on the
stability of a trehalose-releasing enzyme from Arthrobacter sp.
S34, FERM BP-6450, according to the present invention.
FIG. 11 is a figure that shows the influence of pH on the stability
of a trehalose-releasing enzyme from Arthrobacter sp. S34, FERM
BP-6450, according to the present invention.
FIG. 12 is a restriction map of the recombinant DNA pGZ2 according
to the present invention. The bold line shows the nucleotide
sequence from Arthrobacter sp. S34, FERM BP-6450. The oblique arrow
within the bold line shows a nucleotide sequence encoding the
present trehalose-releasing enzyme.
FIG. 13 is a restriction map of the recombinant DNA pGZ3 according
to the present invention. The oblique arrow shows the nucleotide
sequence from Arthrobacter sp. S34, FERM BP-6450.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a non-reducing saccharide-forming
enzyme and a trehalose-releasing enzyme, and a process for
producing a saccharide using either or both of the enzymes. The
wording "non-reducing saccharide-forming enzyme" as referred to in
the present invention represents an enzyme which has an action of
forming a non-reducing saccharide having a trehalose structure as
an end unit from reducing partial starch hydrolysates. The wording
"trehalose-releasing enzyme" as referred to in the present
invention represents an enzyme which specifically hydrolyses a
non-reducing saccharide having a trehalose structure as an end unit
and a glucose polymerization degree of at least 3 to release
trehalose from the rest of the non-reducing saccharide. The wording
"a medium temperature range" as referred to in the present
invention represents a middle temperature range in reaction
temperatures which are conventionally used in producing saccharides
from starch materials by an enzymatic reaction. In most cases of
such processes, different reaction temperatures of about 10.degree.
C. to about 100.degree. C. and a round the temperatures are used.
The nonreducing saccharide-forming enzyme according to the present
invention has an action as such an enzyme and has an optimum
temperature in a medium temperature range, preferably a temperature
range over 40.degree. C. but less than 60.degree. C., and more
preferably it has an optimum pH in an acid pH range in addition to
the optimum temperature. The trehalose-releasing enzyme according
to the present invention has an action as such an enzyme and has an
optimum temperature in a medium temperature range, preferably a
temperature range over 45.degree. C. but below 60.degree. C., and
more preferably it has an optimum pH in an acid pH range in
addition to the optimum temperature. These present enzymes should
not be restricted to their origins and sources.
The activity of the present non-reducing saccharide-forming enzyme
is assayed as follows: One ml of an enzyme solution is added to
four ml of 1.25 w/v % maltopentaose as a substrate in 20 mM
phosphate buffer (pH 6.0), and the mixture solution is incubated at
50.degree. C. for 60 min. The reaction mixture is heated at
100.degree. C. for 10 min to suspend the enzymatic reaction, and
the reaction mixture is precisely diluted by 10 times with
deionized water, followed by determining the reducing power of the
diluted solution on the Somogyi-Nelson's method. As a control, an
enzyme solution, which had been heated at 100.degree. C. for 10 min
to inactivate the enzyme, is treated similarly as above. One unit
activity of the present enzyme is defined as the amount of enzyme
which eliminates the reducing power of that of one .mu.mole of
maltopentaose per minute when determined with the above-mentioned
assay. The optimum temperature of the enzyme as referred to in the
present invention is determined in accordance with the assay; It is
assayed by adjusting the enzymatic reaction temperature at
different temperatures including 50.degree. C., allowing a
prescribed amount of the enzyme to act on the substrate at the
different temperatures according to the assay, and determining the
reduction level of reducing power at the temperatures in accordance
with the assay, followed by comparing the determined reduction
levels one another and determining the optimum temperature of the
present enzyme that showed a maximum temperature.
The activity of the present trehalose-releasing enzyme is assayed
as follows: One ml of an enzyme solution is added to four ml of
1.25 w/v % maltotriosyltrehalose, i.e.,
.alpha.-maltotetraosyl-.alpha.-D-glucoside, as a substrate, in 20
mM phosphate buffer (pH 6.0), and the mixture solution is incubated
at 50.degree. C. for 30 min, followed by suspending the enzymatic
reaction by the addition of the Somogyi copper solution and
assaying the reducing power by the Somogyi-Nelson's method. As a
control, it is similarly assayed using an enzyme solution which has
been inactivated by heating at 100.degree. C. for 10 min. One unit
activity of the present enzyme is defined as the amount of enzyme
which increases the reducing power of one .mu. mole of glucose per
minute when determined with the above-mentioned assay. The optimum
temperature of the enzyme as referred to in the present invention
is determined in accordance with the assay; It is assayed by
adjusting the enzymatic reaction temperature at the different
temperatures including 50.degree. C., allowing a prescribed amount
of the enzyme to act on the substrate at the temperatures according
to the assay, and determining the increased level of reducing power
at the different temperatures in accordance with the assay,
followed by comparing the determined increased levels one another
and determining the optimum temperature of the present enzyme that
showed a maximum temperature.
Explaining the present non-reducing saccharide-forming enzyme based
on the amino acid sequence, the enzyme has the amino acid sequence
of SEQ ID NO:1 as a whole, and has the amino acid sequences of SEQ
ID NOs:2 to 6 as partial amino acid sequences in some cases. In
addition to these enzymes having the whole of the above-identified
amino acid sequences, the present invention includes another types
of enzymes which comprise a part of any one of the amino acid
sequences selected therefrom or which have both the action as the
present non-reducing saccharide-forming enzyme and the
above-identified optimum temperature. Examples of the amino acid
sequences of such enzymes are those which contain, within the amino
acid sequences, a partial amino acid sequence or an amino acid
residue that are related to the expression of the properties of the
present non-reducing saccharide-forming enzyme, and which one or
more amino acids are replaced with different amino acids, added
thereunto and/or deleted therefrom other than the above partial
amino acid sequence or the amino acid residue. Examples of the
amino acid sequences replaced with different amino acids as
referred to in the present invention include those which less than
30% and preferably less than 20% of the amino acid sequences
composing the amino acid sequence of SEQ ID NO:1 are replaced with
another amino acids which have similar properties and structures to
respective ones to be replaced. Examples of groups of such amino
acids are a group of aspartic acid and glutamic acid as acid amino
acids, one of lysine, arginine, and histidine as basic amino acids,
one of asparagine and glutamine as amid-type amino acids, one of
serine and threonine as hydroxyamino acids, and one of valine,
leucine and isoleucine as branched-chain amino acids. Examples of
another amino acid sequences of the present enzyme containing a
part of any one of the amino acid sequences selected from SEQ ID
NOs:1 to 6 are those which might have a substantially similar
stereo-structure to the one of the amino acid sequence of SEQ ID
NO:1, i.e., replacement, deletion and/or addition of amino acid(s)
are introduced into the amino acid sequence of SEQ ID NO:1. The
stereo-structure of proteins is estimable by screening commercially
available databases for stereo-structures of proteins which have
amino acid sequences related to the aiming ones and have revealed
stereo-structures, referencing the screened stereo-structures, and
using commercially available soft wares for visualizing
stereo-structures. The above-identified amino acid sequence of the
present non-reducing saccharide-forming enzyme has a homology of at
least 57%, preferably at least 70%, and more preferably at least
80% to SEQ ID NO:1.
As described above, the non-reducing saccharide-forming enzyme
should not be restricted to a specific origin/source. Examples of
such are those derived from microorganisms, i.e., those of the
genus Arthrobacter, Arthrobacter sp. S34, FERM BP-6450, and its
mutants. The mutants can be obtained by treating in a usual manner
Arthrobacter sp. S34, FERM BP-6450, with known mutagens such as
N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate,
ultraviolet, and transposon; screening the desired mutants capable
of producing a non-reducing saccharide-forming enzyme and having an
optimum temperature at temperatures in a medium temperature range,
and usually at temperatures in the range of over 40.degree. C. but
below 60.degree. C. The enzyme from Arthrobacter sp. S34, FERM
BP-6450, usually has the amino acid sequences of SEQ ID NOs:1 to 6.
Another non-reducing saccharide-forming enzymes from microorganisms
of mutants Arthrobacter sp. S34, FERM BP-6450, and another
microorganisms comprise the whole or a part of any one of the amino
acid sequences of SEQ ID NOs:1 to 6. Concrete examples of another
enzymes include recombinant enzymes which act as the present
non-reducing saccharide-forming enzyme and have an optimum
temperature at temperatures in a medium temperature range, and
usually at temperatures of over 40.degree. C. but below 60.degree.
C. The recombinant enzymes can be obtainable by applying the
recombinant DNA technology for the DNA encoding the present
non-reducing saccharide-forming enzyme, and have the whole or a
part of any one of the amino acid sequences of SEQ ID NOs:1 to
6.
Most of the non-reducing saccharide-forming enzyme according to the
present invention has the following physicochemical properties: (1)
Action Forming a non-reducing saccharide having a trehalose
structure as an end unit from a reducing partial starch
hydrolysates having a degree of glucose polymerization of 3 or
higher; (2) Molecular Weight About 75,000.+-.10,000 daltons on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE); (3) Isoelectric Point (pI) About 4.5.+-.0.5 on
isoelectrophoresis using ampholyte; (4) Optimum Temperature About
50.degree. C. when incubated at pH 6.0 for 60 min; (5) Optimum pH
About 6.0 when incubated at 50.degree. C. for 60 min; (6) Thermal
Stability Stable up to a temperature of about 55.degree. C. when
incubated at pH 7.0 for 60 min; and (7) pH Stability Stable at pHs
of about 5.0 to about 10.0 when incubated at 4.degree. C. for 24
hours.
The present non-reducing saccharide-forming enzyme can be obtained
in a prescribed amount by the later described present process for
producing the same.
The present invention provides a DNA encoding the present
non-reducing saccharide-forming enzyme. Such a DNA is quite useful
in producing the enzyme in the form of a recombinant protein. In
general, the DNA includes those which encode the enzyme
independently of its origin/source. Examples of such a DNA are
those which contain the whole or a part of the nucleotide sequence
of SEQ ID NO:7 or complementary ones thereunto. The DNA comprising
the whole of the nucleotide sequence of SEQ ID NO:7 encodes the
amino acid sequence of SEQ ID NO:1. The DNAs, which contain the
whole or a part of the nucleotide sequence of SEQ ID NO:7, include
those which have an amino acid sequence relating to the expression
of the properties of the present non-reducing saccharide-forming
enzyme, and have a nucleotide sequence corresponding to the amino
acid sequence, and the nucleotide sequence of SEQ ID NO:7
introduced with a replacement, deletion and/or addition of one or
more bases while retaining the nucleotide sequence relating to the
expression of the properties of the present non-reducing
saccharide-forming enzyme. The DNAs according to the present
invention should include those which one or more bases are replaced
with different ones based on the degeneracy of genetic code. Also
the DNAs according to the present invention include those which
comprise the nucleotide sequences that encode the present
non-reducing saccharide-forming enzyme and further comprise
additional one or more another nucleotide sequences selected from
the group consisting of ribosome-binding sequences such as an
initiation codon, termination codon, and Shine-Dalgarno sequence;
nucleotide sequences encoding signal peptides, recognition
sequences for appropriate restriction enzymes; nucleotide sequences
to regulate the expression of genes for promotor and enhancers; and
terminators, all of which are generally used in recombinant DNA
technology for producing recombinant proteins. For example, since a
part of and the whole of the nucleotide sequence of SEQ ID NO:8
function as ribosome-binding sequences, DNAs to which the part of
and the whole of the nucleotide sequence of SEQ ID NO:8 are ligated
upstream of the nucleotide sequences encoding the present
non-reducing saccharide-forming enzyme can be arbitrarily used in
producing the enzyme as a recombinant protein.
As described above, the DNAs encoding the present non-reducing
saccharide-forming enzyme should not be restricted to their
origins/sources, and they are preparable by screening DNAs from
different sources based on hybridization with a DNA comprising a
nucleotide sequence which encodes at least a part of the amino acid
sequence of the enzyme, eg., the amino acid sequence of SEQ ID
NO:1. Actual examples of these sources are microorganisms of the
genus Arthrobacter, and preferably, Arthrobacter sp. S34, FERM
BP-6450, and its mutants, all of which produce the non-reducing
saccharide-forming enzyme. To screen the microorganisms,
conventional methods used in this field for screening or cloning
DNAs such as screening methods of recombinant libraries, PCR
method, and their modified methods. As a result of screening, the
desired DNAs can be obtained by collecting in a usual manner DNAs
confirmed with the expected hybridization. Generally, the DNAs thus
obtained comprise a part of or the whole of the nucleotide sequence
of SEQ ID NO:7. For example, a DNA which comprises the whole of the
nucleotide sequence of SEQ ID NO:7 is generally obtained from
Arthrobacter sp. S34, FERM BP-6450. DNAs comprising a part of the
nucleotide sequence of SEQ ID NO:7 can be obtained by similarly
screening DNAs from microorganisms as sources other than the above
strain, capable of producing the present non-reducing
saccharide-forming enzyme. Such DNAs can be prepared by selecting
DNAs, which encode the enzymes having the properties of the present
enzyme, from DNAs into which have been introduced a replacement,
addition and/or deletion of one or more bases of the
above-mentioned DNAs by using one or more conventional
mutation-introducing methods. The DNAs can be also obtained by
applying conventional chemical syntheses based on the nucleotide
sequence encoding the present non-reducing saccharide-forming
enzyme, e.g., one of SEQ ID NO:7. Once in hand, the DNAs according
to the present invention can be easily amplified to the desired
level by applying or using PCR method and autonomously-replicable
vectors.
The present DNA encoding the non-reducing saccharide-forming enzyme
include those in the form of recombinant DNAs which the DNAs have
been introduced into appropriate vectors. The recombinant DNAs can
be relatively-easily preparable by recombinant DNA technology in
general if only the DNAs are available. Any types of vectors can be
used in the present invention as long as they autonomously
replicable in appropriate hosts. Examples of such vectors are
pUC18, pBluescript II SK(+), pKK223-3, .lamda.gt.lamda.C, etc.,
which use Escherichia coli as a host; pUB110, pTZ4, pC194, .rho.11,
.phi.1, .phi.105, etc., which use microorganisms of the genus
Bacillus; and pHY300PLK, pHV14, TRp7, YEp7, pBS7, etc., which use
two or more microorganisms as hosts. The methods to insert the
present DNA into such vectors in the present invention may be
conventional ones generally used in this field. A gene containing
the present DNA and an autonomously-replicable vector are first
digested with a restriction enzyme and/or ultrasonic disintegrator,
then the resultant DNA fragments and vector fragments are ligated.
The ligation is facilitated by the use of restriction enzymes which
specifically act on the cleavage of the DNA, especially, KpnI,
AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI, SacI, SalI, SmaI,
SpeI, XbaI, XhoI, etc. To ligate DNA fragments and vectors, firstly
they may be annealed if necessary, then subjected to the action of
a DNA ligase in vivo or in vitro. The recombinant DNA thus obtained
can be replicable without substantial limitation in an appropriate
host.
The present DNA encoding the non-reducing saccharide-forming enzyme
further includes transformants which the DNA has been introduced
into appropriate vectors. The transformants can be easily
preparable by introducing the DNA or recombinant DNA obtained in
the above into appropriate hosts to transform them. As the hosts,
microorganisms and cells from plants and animals, which are used
conventionally in this field and chosen depending on the vectors in
the recombinant DNA, can be used. The microorganisms as hosts
include those of the genera Escherichia, Bacillus, and
Arthrobacter, and another actinomycetes, yeasts, fungi, etc. To
introduce the present DNA into these host microorganisms,
conventional competent cell method and protoplast method can be
used. The present DNA, which encodes the non-reducing
saccharide-forming enzyme introduced into the transformants in the
present invention, may be present in a separatory form from
chromosomes or in an incorporated form into chromosomes. The DNA
incorporated into hosts' chromosomes has a character of being
stably retained therein and may be advantageously used in producing
the present recombinant protein.
The present non-reducing saccharide-forming enzyme can be obtained
in a desired amount by a process for producing the enzyme
characterized in that it comprises the steps of culturing
microorganisms capable of producing the enzyme, and collecting the
produced enzyme from the culture. The microorganisms used in the
process can be used independently of the genus or the species as
long as they produce the enzyme. Examples of such microorganisms
are microorganisms of the genus Arthrobacter, Arthrobacter sp. S34,
FERM BP-6450, and mutants thereof, as well as transformants
obtainable by introducing the present DNA encoding the enzyme into
appropriate hosts.
Any nutrient culture media used in culturing the process for
producing the present non-reducing saccharide-forming enzyme can be
used as long as the aforesaid microorganisms grow therein and
produce the enzyme without restriction to a specific nutrient
culture medium. Generally, the nutrient culture media contain
carbon and nitrogen sources, and if necessary minerals may be
added. Examples of the carbon sources are saccharides such as
dextrins, starches, partial starch hydrolysates, glucose, etc., and
are saccharide-containing substances such as molasses and yeast
extracts, and organic acids such as glucuronic acid and succinic
acid. The concentration of the carbon sources is chosen depending
on the types used, usually 30 w/v %, and preferably 15 w/w % or
lower. Examples of the nitrogen sources appropriately used in the
present invention are inorganic-nitrogen-containing substances such
as ammonium salts, nitrate, etc.; organic-nitrogen-containing
substances such as urea, corn steep liquor, casein, peptone, yeast
extract, beef extract, etc. Depending on use, it is selectively
used among inorganic ingredients such as salts of calcium,
magnesium, potassium, sodium, phosphoric acid, manganese, zinc,
iron, copper, molybdenum, cobalt, etc.
The culture conditions used for producing the present enzyme can be
used selectively from appropriate conditions suitable for growing
respective microorganisms used. For example, in the case of using
microorganisms of the genus Arthrobacter including Arthrobacter sp.
S34, FERM BP-6450, the cultivation temperature is usually in the
range of 20-50.degree. C., and preferably 25-37.degree. C.; the
cultivation pH is usually in the range of pH 4-10, and preferably
pH 5-9; and the cultivation time is in the range of 10-150 hours.
With these conditions, the microorganisms are cultured under
aerobic conditions. When used transformants prepared by introducing
into appropriate hosts the present DNA encoding the present
non-reducing saccharide-forming enzyme, the transformants are
cultured under aerobic conditions at conditions selected from the
culture conditions such as the culture temperatures of
20-65.degree. C., the culture pH of 2-9, and the culture time of
1-6 days, although they vary depending on the genus, species,
strains or types of microorganisms and vectors. The cultures thus
obtained generally contain the present enzyme in cell fractions. In
the case of culturing transformants obtained by using as hosts the
microorganisms of the genus Bacillus, the resulting cultures may
contain the present enzyme in supernatant fractions depending on
vectors used to transform the hosts. The content of the present
enzyme in the cultures thus obtained is usually 0.01-1,000 units
per ml of the culture, though it varies depending on the genus,
species or strains of the microorganisms and culture conditions
used.
The present non-reducing saccharide-forming enzyme is collected
from the resulting cultures. The collection method is not
restricted; The present enzyme can be obtained by separating and
collecting any one of fractions of cells and culture supernatants
found with a major activity of the enzyme, and if necessary
subjecting the collected fraction to an appropriate purification
method to collect a purified fraction containing the enzyme. To
separate the fractions of cells and culture supernatants of the
cultures, conventional solid-liquid separation methods such as
centrifugation and filtration using precoat filters and plain- and
hollow fiber-membranes can be arbitrarily used. The desired
fractions are collected from the separated fractions of cells and
culture supernatant. For the fraction of cells, the cells are
disrupted into a cell disruptant which is then separated into a
cell extract and an insoluble cell fraction, followed by collecting
either of the desired fractions. The insoluble cell fraction can be
solubilized by conventional methods, if necessary. As a method to
disrupt cells, any one of techniques of ultrasonication, treatment
with cell-wall-lysing enzymes such as lysozyme and glucanase, and
load of mechanical press can be arbitrarily used. To disrupt cells
the cultures can be directly treated with any one of the above
techniques, and then resulting mixtures are treated with any one of
the above solid-liquid separation methods to collect a liquid
fraction. Thus a cell extract can be arbitrarily obtained.
The methods used for more purifying the present non-reducing
saccharide-forming enzyme include conventional ones to purify
saccharide-related enzymes in general such as salting out,
dialysis, filtration, concentration, gel filtration chromatography,
ion-exchange chromatography, hydrophobic chromatography,
reverse-phase chromatography, affinity chromatography, gel
electrophoresis and, isoelectric point electrophoresis. These
methods can be used in combination depending on purposes. From the
resulting fractions separated by these methods, fractions with a
desired activity assayed by the method for non-reducing
saccharide-forming enzyme are collected to obtain the present
non-reducing saccharide-forming enzyme purified to a desired level.
According to the methods in the later described Examples, the
present enzyme can be purified up to an electrophoretically
homogenous level. As described above, the present method provide
the present non-reducing saccharide-forming enzyme in the form of a
culture, cell fraction, fraction of culture supernatant, cell
disruptant, cell extract, soluble and insoluble cell-fraction,
partially purified enzyme fraction, and purified enzyme fraction.
These fractions may contain another type of the present
trehalose-releasing enzyme. The non-reducing saccharide-forming
enzyme thus obtained can be immobilized in a usual manner before
use. The methods for immobilization are, for example, binding
method to ion exchangers, covalent bonding/adsorption to and on
resins and membranes, and entrapping immobilization method using
high molecular weight substances. The non-reducing
saccharide-forming enzyme thus obtained can be arbitrarily used in
processes for producing saccharides including the later described
present process for producing saccharide. Particularly, since the
present non-reducing saccharide-forming enzyme has an optimum
temperature in a medium temperature range and preferably has an
optimum pH in an acid pH range, it can be advantageously used to
produce saccharides when used in combination with the later
described present trehalose-releasing enzyme, starch-debranching
enzyme having an optimum pH in an acid pH range, and
cyclomaltodextrin glucanotransferase that effectively acts at
medium temperature range.
Explaining the present trehalose-releasing enzyme based on the
amino acid sequence, the enzyme has the amino acid sequence of SEQ
ID NO:9 as a whole, and has the amino acid sequences of SEQ ID
NOs:10 to 16 as partial amino acid sequences in some cases. In
addition to these enzymes having the whole of the above-identified
amino acid sequences, the present invention includes another types
of enzymes which comprise a part of any one of the amino acid
sequences selected therefrom or which have both the action as the
present trehalose-releasing enzyme and the above-identified optimum
temperature. Examples of the amino acid sequences of such enzymes
are those which contain, within the amino acid sequences, a partial
amino acid sequence or an amino acid residue which relate to the
expression of the properties of the present non-reducing
saccharide-forming enzyme, and which one or more amino acids are
replaced with different amino acids, added thereunto and/or deleted
therefrom other than the above partial amino acid sequence or the
amino acid residue. Examples of amino acid sequences replaced with
different amino acids as referred to in the present invention
include those which less than 30% and preferably less than 20% of
the amino acid sequences composing the amino acid sequence of SEQ
ID NO:9 are replaced with another amino acids which have similar
properties and structures to respective ones to be replaced.
Examples of groups of such amino acids are a group of aspartic acid
and glutamic acid as acid amino acids, one of lysine, arginine, and
histidine as basic amino acids, one of asparagine and glutamine as
amid-type amino acids, one of serine and threonine as hydroxyamino
acids, and one of valine, leucine and isoleucine as branched-chain
amino acids. Examples of another amino acid sequences of the enzyme
containing a part of any one of the amino acid sequences selected
from SEQ ID NOs:9 to 16 are those which might have a substantially
similar stereo-structure to the one of the amino acid sequence of
SEQ ID NO:9, i.e., replacement, deletion and/or addition of amino
acid(s) are introduced into the amino acid sequence of SEQ ID NO:9.
The stereo-structure of proteins is estimable by screening
commercially available databases for stereo-structures of proteins
which have amino acid sequences related to the aiming ones and have
revealed stereo-structures, referencing the screened
stereo-structures, and using commercially available soft wares for
visualizing stereo-structures. The above-identified amino acid
sequence of the present trehalose-releasing enzyme has a homology
of at least 60%, preferably at least 70%, and more preferably at
least 80% to SEQ ID NO:9.
As described above, the trehalose-releasing enzyme should not be
restricted to a specific origin/source. Examples of such are those
derived from microorganisms, i.e., those of the genus Arthrobacter,
Arthrobacter sp. S34, FERM BP-6450, and mutants thereof. The
mutants can be obtained by treating in a usual manner Arthrobacter
sp. S34, FERM BP-6450, with known mutagens such as
N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate,
ultraviolet, and transposon; screening the desired mutants capable
of producing a non-reducing saccharide-forming enzyme and having an
optimum temperature at temperatures in a medium temperature range,
and usually at temperatures in the range of over 45.degree. C. but
below 60.degree. C. The enzyme from Arthrobacter sp. S34, FERM
BP-6450, usually has the amino acid sequences of SEQ ID NOs:9 to
16. Another non-reducing saccharide-forming enzymes from
microorganisms of mutants Arthrobacter sp. S34, FERM BP-6450, and
another microorganisms comprise the whole or a part of any one of
the amino acid sequences of SEQ ID NOs:9 to 16. Concrete examples
of another enzymes include recombinant enzymes which act as the
present trehalose-releasing enzyme and have an optimum temperature
at temperatures in a medium temperature range, and usually at
temperatures of over 45.degree. C. but below 60.degree. C. The
recombinant enzymes can be obtainable by applying the recombinant
DNA technology for the DNA encoding the present trehalose-releasing
enzyme, and have the whole or a part of any one of the amino acid
sequences of SEQ ID NOs:9 to 16.
Most of the trehalose-releasing enzyme according to the present
invention has the following physicochemical properties: (1) Action
Specifically hydrolyses a non-reducing saccharide having a
trehalose structure as an end unit to release trehalose from the
rest of the non-reducing saccharide; (2) Molecular weight About
62,000.+-.5,000 daltons on sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDSPAGE); (3) Isoelectric point (PI) About
4.7.+-.0.5 on isoelectrophoresis using ampholyte; (4) Optimum
temperature About 50.degree. C. to about 55.degree. C. when
incubated at pH 6.0 for 30 min; (5) Optimum pH About 6.0 when
incubated at 50.degree. C. for 30 min; (6) Thermal stability Stable
up to a temperature of about 50.degree. C. when incubated at pH 7.0
for 60 min; and (7) pH Stability Stable at pHs of about 4.5 to
about 10.0 when incubated at 4.degree. C. for 24 hours.
The present trehalose-releasing enzyme can be obtained in a
prescribed amount by the later described present process for
producing the same.
The present invention provides a DNA encoding the present
trehalose-releasing enzyme. Such a DNA is quite useful in producing
the enzyme in the form of a recombinant protein. In general, the
DNA includes those which encode the enzyme independently of its
origin/source. Examples of such a DNA are those which contain the
whole or a part of the nucleotide sequence of SEQ ID NO:17 or
complementary ones thereunto. The DNA comprising the whole of the
nucleotide sequence of SEQ ID NO:17 encodes the amino acid sequence
of SEQ ID NO:9. The DNAs, which contain the whole or a part of the
nucleotide sequence of SEQ ID NO:17, include those which have a
nucleotide sequence corresponding to an amino acid sequence
relating to the expression of the properties of the present
non-reducing saccharide-forming enzyme, and have the nucleotide
sequence of SEQ ID NO:17 introduced with a replacement, deletion
and/or addition of one or more bases while retaining the nucleotide
sequence relating to the expression of the properties of the
present trehalose-releasing enzyme. The DNAs according to the
present invention should include those which one or more bases are
replaced with different ones based on the degeneracy of genetic
code. Also the DNAs according to the present invention include
those which comprise the nucleotide sequences that encode the
present trehalose-releasing enzyme and further comprise additional
one or more another nucleotide sequences selected from the group
consisting of ribosome-binding sequences such as an initiation
codon, termination codon, and Shine-Dalgarno sequence; nucleotide
sequences encoding signal peptides, recognition sequences for
appropriate restriction enzymes; nucleotide sequences to regulate
the expression of genes for promotor and enhancers; and
terminators, all of which are generally used in recombinant DNA
technology for producing recombinant proteins. For example, since a
part of and the whole of the nucleotide sequence of SEQ ID NO:8
function as ribosome-binding sequences, DNAs to which the part of
and the whole of the nucleotide sequence of SEQ ID NO:8 are ligated
upstream of the nucleotide sequences encoding the present
trehalose-releasing enzyme can be arbitrarily used in producing the
enzyme as a recombinant protein.
As described above, the DNAs encoding the present
trehalose-releasing enzyme should not be restricted to their
origins/sources, and they are preparable by screening DNAs from
different sources based on hybridization with a DNA comprising a
nucleotide sequence which encodes at least a part of the amino acid
sequence of the enzyme, eg., the amino acid sequence of SEQ ID
NO:9. Actual examples of these sources are microorganisms of the
genus Arthrobacter, and preferably, Arthrobacter sp. S34, FERM
BP-6450, and its mutants, all of which produce the non-reducing
saccharide-forming enzyme. To screen the microorganisms,
conventional methods used in this field for screening or cloning
DNAs such as screening methods of recombinant libraries, PCR
method, and their modified methods. As a result of screening, the
desired DNAs can be obtained by collecting in a usual manner DNAs
confirmed with the expected hybridization. Generally, the DNAs thus
obtained comprise a part of or the whole of the nucleotide sequence
of SEQ ID NO:17. For example, a DNA which comprises the whole of
the nucleotide sequence of SEQ ID NO:17 is generally obtained from
Arthrobacter sp. S34, FERM BP-6450. DNAs comprising a part of the
nucleotide sequence of SEQ ID NO:17 can be obtained by similarly
screening DNAs from microorganisms as sources other than the above
strain, capable of producing the trehalose-releasing enzyme. Such
DNAs can be prepared by selecting DNAs, which encode the enzymes
having the properties of the enzyme, from DNAs into which have been
introduced a replacement, addition and/or deletion of one or more
bases of the above-mentioned DNAs by using one or more conventional
mutation-introducing methods. The DNAs can be also obtained by
applying conventional chemical syntheses based on the nucleotide
sequence encoding the present trehalose-releasing enzyme, e.g., one
of SEQ ID NO:17. Once in hand, the DNAs according to the present
invention can be easily amplified to the desired level by applying
or using PCR method and autonomously-replicable vectors.
The present DNA encoding the trehalose-releasing enzyme include
those in the form of recombinant DNAs which the DNAs have been
introduced into appropriate vectors. The recombinant DNAs can be
relatively-easily preparable by recombinant DNA technology in
general if only the DNAs are available. Any types of vectors can be
used in the present invention as long as they autonomously
replicable in appropriate hosts. Examples of such vectors are
pUC18, pBluescript II SK(+), pKK223-3, .lamda.gt.lamda.C, etc.,
which use Escherichia coli as a host; pUB110, pTZ4, pC194, .rho.11,
.phi.1, .phi.105, etc., which use microorganisms of the genus
Bacillus; and pHY300PLK, pHV14, TRp7, YEp7, pBS7, etc., which use
two or more microorganisms as hosts. The methods to insert the
present DNA into such vectors in the present invention may be
conventional ones generally used in this field. A gene containing
the present DNA and an autonomously-replicable vector are first
digested with a restriction enzyme and/or ultrasonic disintegrator,
then the resultant DNA fragments and vector fragments are ligated.
The ligation is facilitated by the use of restriction enzymes which
specifically act on the cleavage of the DNA, especially, KpnI,
AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI, SacI, SalI, SmaI,
SpeI, XbaI, XhoI, etc. To ligate DNA fragments and vectors, firstly
they may be annealed if necessary, then subjected to the action of
a DNA ligase in vivo or in vitro. The recombinant DNA thus obtained
can be replicable without substantial limitation in an appropriate
host.
The present DNA encoding the trehalose-releasing enzyme further
includes transformants which the DNA has been introduced into
appropriate vectors. The transformants can be easily preparable by
introducing the DNA or recombinant DNA obtained in the above into
appropriate hosts to transform them. As the hosts, microorganisms
and cells from plants and animals, which are used conventionally in
this field and chosen depending on the vectors in the recombinant
DNA, can be used. The microorganisms as hosts include those of the
genera Escherichia, Bacillus, and Arthrobacter, and another
actinomycetes, yeasts, fungi, etc. To introduce the present DNA
into these host microorganisms, conventional competent cell method
and protoplast method can be used. The present DNA, which encodes
the trehalose-releasing enzyme introduced into the transformants in
the present invention, may be present in a separatory form from
chromosomes or in an incorporated form into chromosomes. The DNA
incorporated into hosts' chromosomes has a character of being
stably retained therein and may be advantageously used in producing
the present recombinant protein.
The aforesaid techniques used for obtaining the present DNAs
including recombinant DNAs and transformants, and the techniques
for obtaining the DNAs and recombinant proteins are commonly used
in the art; For example, J. Sumbruck et al. in "Molecular Cloning A
Laboratory Manual", 2nd edition, published by Cold Spring Harbor
Laboratory Press (1989), discloses in detail methods for obtaining
desired DNAs and applications for production use of the obtained
DNAs. For example, Japanese Patent No. 2,576,970 discloses a method
for stabilizing a transformed DNA, which uses as a host a
microorganism defective in an aiming gene. Japanese Patent Kokai
No. 157,987/88 discloses a vector which effectively expresses an
aiming DNA in microorganisms of the genus Bacillus. Japanese Patent
Kohyo No. 502,162/93 discloses a method for stably introducing a
desired DNA into a bacterial chromosome. Japanese Patent Kohyo No.
506,731/96 discloses an efficient production method of a starch
hydrolysing enzyme, using recombinant DNA technology. Japanese
Patent Kohyo Nos. 500,543/97 and 500,024/98 disclose a host-vector
system using fungi for efficient production of recombinant
proteins. These methods conventionally used in the art are
arbitrarily applicable for the present invention.
In the art, when the desired DNAs are available by the above
methods, there have been commonly provided transformants which the
DNAs are introduced into appropriate plants and animals, i.e.,
transgenic plants and animals. The present DNA, which encodes the
non-reducing saccharide-forming enzyme and the trehalose-releasing
enzyme in the form of a DNA introduced into appropriate hosts, also
includes the transgenic plants and animals. To obtain the
transgenic animals, it is obtained as a whole by a process
comprising the DNA which encodes either of the present enzymes
alone or together with other desired DNA such as a promotor and
enhancer into an appropriate vector selected depending on the
species of the host animal, introducing the resulting recombinant
DNA into a fertilized egg or embryonic stem cell from the host
animal by a method such as micro-injection and electroporation, or
by an infection method using recombinant viruses containing the
recombinant DNA. Examples of the host animals are conventional
experimental rodents such as mice, rats, and hamsters; and mammals
conventionally used as domestic animals such as goats, sheep, pigs,
and cows, all of which have an advantage of being bred easily. The
resulting cells introduced with the DNA are transplanted in uterine
tube or uterus of a pseudopregnancy female animal of the same
species as the cells. Thereafter, transgenic animals, which have
been introduced with the DNA encoding the present enzymes by
applying hybridization or PCR method, are obtained from newborns in
a natural or cesarean sectional manner. Thus the present DNA in the
form of a transgenic animal can be obtained. Referring to
transgenic animals, they are disclosed in detail in
"Jikken-Igaku-Bessatsu-Shin-Idennshi-Kogaku-Handbook" (Handbook of
Genetic Engineering), pp. 269-283 (1996), edited by Masami
MATSUMURA, Hiroto OKAYAMA, and Tadashi YAMAMOTO, published by
Yodosha Co., Ltd., Tokyo, Japan. The method for obtaining
transgenic plants comprises, for example, providing a plasmid as a
vector of a microorganism of the genus Agrobacterium infectious to
plants, introducing the DNA encoding either of the present enzymes
into the vector, and either introducing the resulting recombinant
DNA into plant bodies or protoplasts, or coating heavy metal
particles with a DNA including nucleotide sequence encoding either
of the present enzymes and directly injecting the coated particles
into plant bodies or protoplasts using a particle gun. Although
various types of plants can be used as host plants, they generally
include edible plants such as potato, soybean, wheat, burley, rice,
corn, tomato, lettuce, alfalfa, apple, peach, melon, etc. By
applying hybridization or PCR method for the above transformed
plant bodies and protoplasts, transformants containing the desired
DNA are selected. The transformed protoplasts can be regenerated
into plant bodies as the present DNA in the form of transgenic
plants. The techniques of transgenic plants are generally disclosed
in Genetic Engineering, edited by Jane K. Setlow, published by
Plenum Publishing Corporation, NY, USA, Vol. 16, pp. 93-113 (1994).
The DNA in the form of the aforesaid transgenic animals and plants
can be used as sources of the present non-reducing
saccharide-forming enzyme and/or trehalose-releasing enzyme, and
used as edible plants and animals which contain trehalose or
non-reducing saccharide having a trehalose structure.
The present trehalose-releasing enzyme can be obtained in a desired
amount by the present process for producing the enzyme which is
characterized in that it comprises culturing a microorganism
capable of producing the enzyme in a nutrient culture medium, and
collecting the produced enzyme from the resulting culture. Any
microorganisms can be used in the present process independently of
their genus and species as long as they produce the present
trehalose-releasing enzyme. Examples of such microorganisms are
those of the genus Arthrobacter, Arthrobacter sp. S34, FERM
BP-6450, and mutants thereof, as well as transformants obtainable
by introducing the present DNA encoding the enzyme into appropriate
host microorganisms.
Any nutrient culture media for culturing the process for producing
the present trehalose-releasing enzyme can be used as long as the
aforesaid microorganisms grow therein and produce the enzyme
without restriction to a specific nutrient culture medium.
Generally, the nutrient culture media contain carbon and nitrogen
sources, and if necessary minerals may be added. Examples of the
carbon sources are saccharides such as dextrins, starches, partial
starch hydrolysates, glucose, etc., and are saccharide-containing
substances such as molasses and yeast extracts, and organic acids
such as glucuronic acid and succinic acid. The concentration of the
carbon sources is chosen depending on the types used, usually 30
w/v %, and preferably 15 w/w % or lower. Examples of the nitrogen
sources appropriately used in the present invention are
inorganic-nitrogen-containing substances such as ammonium salts,
nitrate, etc.; organic-nitrogen-containing substances such as urea,
corn steep liquor, casein, peptone, yeast extract, beef extract,
etc. Depending on use, it is selectively used among inorganic
ingredients such as salts of calcium, magnesium, potassium, sodium,
phosphoric acid, manganese, zinc, iron, copper, molybdenum, cobalt,
etc.
The culture conditions used for producing the present
trehalose-releasing enzyme can be used selectively from appropriate
conditions suitable for growing respective microorganisms used. For
example, in the case of using microorganisms of the genus
Arthrobacter including Arthrobacter sp. S34, FERM BP-6450, the
cultivation temperature is usually in the range of 20-50.degree.
C., and preferably 25-37.degree. C.; the cultivation pH is usually
in the range of pH 4-10, and preferably pH 5-9; and the cultivation
time is in the range of 10-150 hours. With these conditions, the
microorganisms are cultured under aerobic conditions. When used
transformants prepared by introducing into appropriate hosts the
present DNA encoding the trehalose-releasing enzyme, the
transformants are cultured under aerobic conditions at conditions
selected from the culture conditions such as the culture
temperatures of 20-65.degree. C., the culture pH of 2-9, and the
culture time of 1-6 days, although they vary depending on the
genus, species, strains or types of microorganisms and vectors. The
cultures thus obtained generally contain the enzyme in cell
fractions. In the case of culturing transformants obtained by using
as hosts the microorganisms of the genus Bacillus, the resulting
cultures may contain the enzyme in supernatant fractions depending
on vectors used to transform the hosts. The content of the enzyme
in the cultures thus obtained is usually 0.01-3,000 units per ml of
the culture, though it varies depending on the genus, species or
strains of the microorganisms and culture conditions used.
The present trehalose-releasing enzyme is collected from the
resulting cultures. The collection method is not restricted; The
enzyme can be obtained by separating and collecting any one of
fractions of cells and culture supernatants found with a major
activity of the enzyme, and if necessary subjecting the collected
fraction to an appropriate purification method to collect a
purified fraction containing the enzyme. To separate the fractions
of cells and culture supernatants of the cultures, conventional
solid-liquid separation methods such as centrifugation and
filtration using precoat filters and plain- and hollow
fiber-membranes can be arbitrarily used. The desired fractions are
collected from the separated fractions of cells and culture
supernatant. For the fraction of cells, the cells are disrupted
into a cell disruptant which is then separated into a cell extract
and an insoluble cell fraction, followed by collecting either of
the desired fractions. The insoluble cell fraction can be
solubilized by conventional methods, if necessary. As a method to
disrupt cells, any one of techniques of ultrasonication, treatment
with cell-wall-lysing enzymes such as lysozyme and glucanase, and
load of mechanical press can be arbitrarily used. To disrupt cells
the cultures can be directly treated with any one of the above
techniques, and then resulting mixtures are treated with any one of
the above solid-liquid separation methods to collect a liquid
fraction. Thus a cell extract can be arbitrarily obtained.
The methods used for more purifying the present trehalose-releasing
enzyme include conventional ones to purify saccharide-related
enzymes in general such as salting out, dialysis, filtration,
concentration, gel filtration chromatography, ion-exchange
chromatography, hydrophobic chromatography, reverse-phase
chromatography, affinity chromatography, gel electrophoresis and,
isoelectric point electrophoresis. These methods can be used in
combination depending on purposes. From the resulting fractions
separated by these methods, fractions with a desired activity
assayed by the method for trehalose-releasing enzyme are collected
to obtain the enzyme purified to a desired level. According to the
methods in the later described Examples, the present enzyme can be
purified up to an electrophoretically homogenous level. As
described above, the present method provide the present
trehalose-releasing enzyme in the form of a culture, cell fraction,
fraction of culture supernatant, cell disruptant, cell extract,
soluble and insoluble cell-fraction, partially purified enzyme
fraction, and purified enzyme fraction. These fractions may contain
another type of the present non-reducing saccharide-forming enzyme.
The present trehalose-releasing enzyme thus obtained can be
immobilized in a usual manner before use. The methods for
immobilization are, for example, binding method to ion exchangers,
covalent bonding/adsorption to and on resins and membranes, and
entrapping immobilization method using high molecular weight
substances. The trehalose-releasing enzyme thus obtained can be
arbitrarily used in processes for producing saccharides including
the later described present process for producing saccharide.
Particularly, since the trehalose-releasing enzyme has an optimum
temperature in a medium temperature range and preferably has an
optimum pH in an acid pH range, it can be advantageously used to
produce saccharides when used in combination with the later
described present trehalose-releasing enzyme, starch-debranching
enzyme having an optimum pH in an acid pH range, and
cyclomatodextrin glucanotransferase that effectively acts at
temperatures in a medium temperature range.
The present invention provides a process for producing saccharides
comprising non-reducing saccharides by using the aforesaid present
enzymes; the process comprising the steps of allowing the
non-reducing saccharide-forming enzyme and/or the
trehalose-releasing enzyme to act on reducing partial starch
hydrolysates to form non-reducing saccharides, and collecting the
resulting non-reducing saccharides or saccharide compositions with
a lesser reducibility. In the process, the use of one or more
another types of non-reducing saccharide-forming enzymes and
trehalose-releasing enzymes other than the present enzymes, and
other saccharide-related enzymes should not be excluded from the
present invention. The reducing partial starch hydrolysates used in
the process can be used independently of their origins/sources. The
non-reducing saccharides as referred to in the present invention
include non-reducing saccharides in general such as trehalose and
those having a trehalose structure.
The reducing partial starch hydrolysates used in the present
process for producing saccharides can be obtained, for example, by
liquefying starches or amylaceous substances by conventional
methods. The starches include terrestrial starches such as corn
starch, rice starch, and wheat starch; and subterranean starches
such as potato starch, sweet potato starch, and tapioca starch. To
liquefy these starches, they are generally suspended in water into
starch suspensions, preferably, those with a concentration of at
least 10 w/w %, and more preferably those with a concentration of
about 20 to about 50 w/w %, and treated with mechanical, acid
and/or enzymatic treatments. Relatively-lower degree of
liquefaction is satisfactorily used, preferably, DE (dextrose
equivalent) of less than 15, and more preferably DE of less than
10. When liquefied with acids, the starches are treated with
hydrochloric acid, phosphoric acid, oxalic acid, etc., and then the
resulting mixtures are neutralized with calcium carbonate, calcium
oxide, sodium carbonate, etc., to desired pHs before use. To
liquefy the starches with enzymes, .alpha.-amylase, particularly,
and thermostable liquefying .alpha.-amylase are satisfactorily
used. The liquefied starches thus obtained can be further subjected
to the action of .alpha.-amylase, maltotriose-forming amylase,
maltotetraose-forming amylase, maltopentaose-forming amylase,
maltohexaose-forming amylase, etc., and the resulting reaction
mixtures can be used as the reducing partial starch hydrolysates.
The properties of the starch-related enzymes are described in
detail in Handbook of Amylases and Related Enzymes, pp. 18-81, and
pp. 125-142 (1988), published by Pergamon Press.
The reducing partial starch hydrolysates thus obtained are
subjected to the action of the present non-reducing
saccharide-forming enzyme and/or trehalose-releasing enzyme, and if
necessary further subjected to the action of one or more
starch-related enzymes such as .alpha.-amylase, .beta.-amylase,
glucoamylase, starch debranching enzymes such as isoamylase and
pullulanase, cyclomaltodextrin glucanotransferase,
.alpha.-glucosidase, and .beta.-fructofuranosidase. Conditions used
for enzymatic reactions are those suitable for enzymes used;
Usually they are selected from pHs 4-10 and temperatures of
20-70.degree. C., and preferably pHs 5-7 and temperatures of
30-60.degree. C. Particularly, non-reducing saccharides can be
effectively produced by enzymatic reactions at temperatures in a
medium temperature range, i.e., temperatures of over 40.degree. C.
but below 60.degree. C. or over 45.degree. C. but below 60.degree.
C., and pHs of slight acid or acid pH conditions. The order of
allowing the enzymes to act on reducing partial starch hydrolysates
is not restricted; one proceeds or follows another one, or plural
enzymes can be arbitrarily allowed to act on substrates
simultaneously.
The amount of enzymes is appropriately set depending on enzymatic
conditions and reaction times, and final uses of non-reducing
saccharides or less-reducible saccharide compositions containing
thereof. For the present non-reducing saccharide-forming enzyme and
trehalose-releasing enzyme, the former is used in an amount of
about 0.01 to about 100 units/g solid of reducing partial starch
hydrolysates, and the latter is used in an amount of about 1 to
about 10,000 units/g solid of reducing partial starch hydrolysates.
Cyclomatodextrin glucanotransferase is used in an amount of about
0.05 to about 500 units/g reducing partial starch hydrolysates,
d.s.b. The reaction mixtures obtained with these enzymes usually
contain trehalose, .alpha.-glucosyltrehalose,
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, or
.alpha.-maltopentaosyltrehalose. In the above process, when used in
combination, the present non-reducing saccharide-forming enzyme and
trehalose-releasing enzyme along with a starch debranching enzyme
and cyclomatodextrin glucanotransferase characteristically more
produce a large amount of trehalose and a relatively-lower
molecular weight of non-reducing saccharide having a trehalose
structure.
From the resulting reaction mixtures, non-reducing saccharides and
saccharide compositions with a lesser reducibility are collected.
In these production steps, conventionally used processed for
saccharides can be appropriately selected. The resulting reaction
mixtures are subjected to filtration and centrifugation to remove
insoluble substances, and then the resultant solutions are purified
by decoloration with an activated charcoal, desalted with ion
exchangers in H-- and OH-form, and concentrated into syrupy
products. If necessary, the syrupy products can be further purified
into non-reducing saccharides with a relatively-high purity; In the
purification, one or more methods, for example, column
chromatographic fractionations such as ion-exchange column
chromatography, column chromatography using an activated charcoal
or a silica gel; separatory sedimentation using organic acids such
as acetone and alcohol; separation using membranes with an
appropriate separability; and alkaline treatments to decompose and
remove the remaining reducing saccharides. In particular,
ion-exchange column chromatography can be suitably used in the
present invention as an industrial-scale preparation of the object
saccharides. Non-reducing saccharides with an improved purity can
be arbitrary prepared by, for example, column chromatography using
a strongly-acid cation exchange resin as described in Japanese
Patent Kokai Nos. 23,799/83 and 72,598/83 to remove concomitant
saccharides. In this case, any of fixed-bed, moving bed, and
semi-moving methods can be employed.
If necessary, the resulting non-reducing saccharides or a
relatively-low reducing saccharides containing the non-reducing
saccharides can be hydrolyzed by amylases such as .alpha.-amylase,
.beta.-amylase, glucoamylase and .alpha.-glucosidase to control
their sweetness and reducing power or to lower their viscosity; and
the products thus obtained can be further treated with processings
where the remaining reducing saccharides are hydrogenated into
sugar alcohols to diminish their reducing powder. Particularly,
trehalose can be easily prepared by allowing glucoamylase or
.alpha.-glucosidase to act on the non-reducing saccharides or
relatively-low reducing saccharides containing the non-reducing
saccharides. A high trehalose content fraction is obtainable by
allowing glucoamylase or .alpha.-glucosidase to act on these
saccharides to form a mixture of trehalose and glucose, and
subjecting the mixture to the aforesaid purification methods such
as column chromatography using ion exchangers to remove glucose.
The high trehalose content fraction can be arbitrary purified and
concentrated into a syrupy product. If necessary, the syrupy
product can be concentrated into a supersaturated solution,
followed by crystallizing hydrous or anhydrous crystalline
trehalose and recovering the resultant crystal.
To produce hydrous crystalline trehalose, an about 65-90 w/w %
solution of trehalose with a purity of about 60 w/w % or higher is
placed in a crystallizer, and if necessary in the presence of
0.1-20 w/v % seed crystal, gradually cooled while stirring at a
temperature of 95.degree. C. or lower, and preferably at a
temperature of 10-90.degree. C. to obtain a massecuite containing
hydrous crystalline trehalose. Continuous crystallization method to
effect crystallization under concentrating conditions in vacuo can
be arbitrarily used.
Conventional methods such as separation, block pulverization,
fluidized-bed granulation, and spray drying can be employed in the
invention to prepare from the massecuite hydrous crystalline
trehalose or crystalline saccharides containing the trehalose
crystal.
In the case of separation, massecuites are usually subjected to a
basket-type centrifuge to separate hydrous crystalline trehalose
from a mother liquor, and if necessary the hydrous crystalline
trehalose is washed by spraying with a small amount of cold water
to facilitate the preparation of hydrous crystalline trehalose with
a higher purity. In the case of spray drying, crystalline
saccharides with no or substantially free of hygroscopicity are
easily prepared by spraying massecuites with a concentration of
70-85 w/w %, on a dry solid basis (d.s.b.), and a crystallinity of
about 20-60%, d.s.b., from a nozzle by a high-pressure pump; drying
the resultant products with air heated to 60-100.degree. C. which
does not melt the resultant crystalline powders; and aging the
resultant powders for about 1 to about 20 hours while blowing
thereto air heated to 30-60.degree. C. In the case of block
pulverization, crystalline saccharides with no or substantially
free of hygroscopicity are easily prepared by allowing massecuites
with a moisture content of 10-20 w/w % and a crystallinity of about
10-60%, d.s.b., to stand for about 0.1 to about 3 days to
crystallize and solidify the whole contents into blocks; and
pulverizing or cutting the resultant blocks.
To produce anhydrous crystalline trehalose, the hydrous crystalline
trehalose obtained in the above is dried at a normal or reduced
pressure at temperatures of 70-160.degree. C., and preferably at
80-100.degree. C.; or a relatively-high concentration and content
trehalose solution with a moisture content of less than 10% is
placed in a crystallizer, stirred in the presence of a seed crystal
at temperatures of 50-160.degree. C., and preferably 80-140.degree.
C. to produce a massecuite containing anhydrous crystalline
trehalose, and treating the massecuite with methods such as block
pulverization, fluidized-bed granulation, and spray drying under
relatively-high temperatures and drying conditions.
The non-reducing saccharides or saccharide composition, containing
thereof with a relatively-low reducibility, thus obtained are low
in reducibility and satisfactory in stability; they do not become
browning, form indisagreeable smell, and deteriorate the following
another materials when mixed and processed with another materials,
for example, amino-acid-containing substances such as amino acids,
oligopeptides, and proteins. Even with a relatively-low
reducibility, the above-identified saccharides have a
relatively-low viscosity, and those with a relatively-low average
glucose polymerization degree have a relatively-high quality and
sweetness. These saccharides can be arbitrarily used in the fields
of foods, cosmetics, and pharmaceuticals, etc., as disclosed in
Japanese Patent Kokai Nos. 66,187/96, 66,188/96, 73,482/96,
73,506/96, 73,504/96, 336,363/96, 9,986/97, 154,493/97, 252,719/97,
66,540/98, and 168,093/98; and Japanese Patent Application Nos.
236,441/97, 256,219/97, 268,202/97, 274,962/97, 320,519/97,
338,294/97, 55,710/98, 67,628/98, 134,553/98 and 214,375/98, which
were all applied for by the same applicant as the present
applicant.
The following examples describe the present invention in more
detail:
EXAMPLE 1
Microorganism Capable of Producing Non-reducing Saccharide-forming
Enzyme and Trehalose-releasing Enzyme
The present inventors widely screened soils to isolate a
microorganism capable of producing non-reducing saccharide-forming
enzyme and trehalose-releasing enzyme. As a result, they isolated a
microorganism with such a property from a soil in Ako, Hyogo,
Japan, and identified the microorganisms in accordance with the
method as described in "Biseibutsu-no-Bunrui-to-Dotei"
(Classification and Identification of Microorganisms), edited by
Takeji Hasegawa, published by Japan Scientific Societies Press,
Tokyo, Japan (1985). The results were as follows:
Results on Cell Morphology (1) Characteristics of cells when
incubated at 37.degree. C. in nutrient agar broth Usually existing
a rod form of 0.4-0.5.times.0.8-1.2 .mu.m; Existing in a single
form but uncommonly existing in a polymorphic form; Free of
motility; Asporogenic; Non-acid fast; and Gram stain: Positive. (2)
Characteristics of cells when incubated at 37.degree. C. in EYG
nutrient agar Exhibiting a growth cycle of rods and cocci.
Results on Cultural Property (1) Characteristics of colony formed
when incubated at 37.degree. C. in nutrient agar broth plate Shape:
Circular colony having a diameter of about 1-2 mm after 2-days
incubation; Rim: Entire; Projection: Convex; Gloss: Moistened
gloss; Surface: Plain; and Color: Semi-transparent or cream. (2)
Characteristics of colony formed when incubated at 37.degree. C. in
nutrient agar broth slant Growth: Satisfactory; and Shape:
Thread-like. (3) Characteristics of colony formed when incubated at
37.degree. C. in agar slant with yeast extract and peptone Growth:
Satisfactory; and Shape: Thread-like. (4) Characteristics of colony
formed when stab-cultured at 27.degree. C. in nutrient gelatin
broth Not liquefying gelatin.
Results on Physiological Properties (1) Methyl red test: Negative
(2) VP-test: Positive (3) Formation of indole: Negative (4)
Formation of hydrogen sulfide: Negative (5) Hydrolysis of starch:
Positive (6) Liquefaction of gelatin: Negative (7) Utilization of
citric acid: Positive (8) Utilization of inorganic nitrogen source:
Utilizing nitrate but not ammonium salts (9) Formation of pigment:
Non (10) Urease: Negative (11) Oxidase: Negative (12) Catalase:
Positive (13) Growth range: Growing at pHs of 4.5-8.0 and
temperatures of 20-50.degree. C.; and Optimum temperatures of
30-45.degree. C. (14) Oxygen requirements: Aerobic (15) Utilization
of carbon sources L-Arabinose: Assimilated D-Glucose: Assimilated
D-Fructose: Not assimilated D-Galactose: Not assimilated
L-Rhamnose: Not assimilated D-Xylose: Not assimilated D-Mannose:
Assimilated Raffinose: Not assimilated Trehalose: Not assimilated
Sucrose: Not assimilated Maltose: Not assimilated Lactose: Not
assimilated D-Dulcitol: Not assimilated D-Mannitol: Not assimilated
Gluconic acid: Assimilated Succinic acid: Assimilated Nicotinic
acid: Not assimilated L-Maleic acid: Assimilated Acetic acid:
Assimilated Lactic acid: Assimilated (16) Acid formation from
sugars L-Arabinose: Slightly formed D-Glucose: Slightly formed
D-fructose: Not formed D-Galactose Slightly formed L-Rhamnose
Slightly formed D-Xylose: Slightly formed Glycerol: Slightly formed
Raffinose: Not formed Trehalose: Slightly formed Sucrose: Slightly
formed Maltose: Slightly formed Lactose: Not formed (17)
Utilization of amino acid Not utilizing sodium L-glutamate, sodium
L-aspartate, L-histidine and L-arginine, (18) Decarboxylase test on
amino acid Negative against L-lysine, L-ornithine and L-arginine.
(19) DNase: Negative (20) N-Acyl type of cell wall: Acetyl (21)
Main diamino acid of cell wall: Lysine (22) Mol % of guanine (G)
plus cytosine (C) of DNA: 71.2%
These bacteriological properties were compared with those of known
microorganisms with reference to Bergey's Manual of Systematic
Bacteriology, Vol. 2 (1984). As a result, it was revealed that the
microorganism was identified as a novel one of the genus
Arthrobacter. Based on the results, the present inventors named
this microorganism "Arthrobacter sp. S34". The microorganisms was
deposited and accepted on Aug. 6, 1998, under the accession number
of FERM BP-6450 in and by the Patent Microorganism Depository,
National Institute of Bioscience and Human-Technology Agency of
Industrial Science & Technology, Ministry of International
Trade & industry, 1-3, Higashi, 1 chome, Tsukuba-shi,
Ibaraki-ken 305-8566, Japan.
The homology of DNA between the identified microorganism and
type-strains of the genus Arthrobacter, deposited in American Type
Culture Collection (ATCC), an international depository of
microorganism in USA, was examined in accordance with the DNA-DNA
hybridization method in Bergey's Manual of Systematic Bacteriology,
Vol. 1 (1984). Twelve type-strains shown in Table 1 in the below
were respectively cultured in a usual manner, and proliferated
cells were collected from the resulting cultures. Arthrobacter sp.
S34, FERM BP-6450, was cultured by the seed culture method in the
later described Example 2-1, followed by collecting the
proliferated cells. According to conventional method, DNAs were
obtained from each type-strain of microorganisms, two micrograms
aliquots of the DNAs were digested with a restriction enzyme, Pst
I. The resulting digested mixtures were respectively spotted on
"Hybond-N+", a nylon membrane commercialized by Amersham
International, Arlington Heights, Ill., USA, and in a usual manner,
treated with alkali, neutralized, and dried to fix the DNAs on the
nylon membrane. One microgram of the DNA obtained from Arthrobacter
sp. S34, FERM BP-6450, was provided and digested with Pst I. Using
[.alpha.-.sup.32P] dCTP commercialized by Amersham International,
Arlington Heights, Ill., USA, and "READY-TO-GO DNA-LABELLING KIT",
a DNA-labelling kit commercialized by Pharmacia LKB Biotechnology
AB, Uppsala, Sweden, the digestant was labelled with an isotope to
obtain a probe. The probe and the above DNA fixed on nylon film
were hybridized for two hours under shaking conditions at
65.degree. C. in "RAPID HYBRIDIZATION BUFFER", a buffer for
hybridization commercialized by Amersham Corp., Div., Amersham
International, Arlington Heights, Ill., USA. The nylon film after
hybridization was washed in a usual manner, dried and subjected to
autoradiography in a usual manner. Signals of hybridization
observed on radiography were analyzed on "IMAGE MASTER", an image
analyzing system commercialized by Pharmacia LKB Biotechnology AB,
Uppsala, Sweden, followed by expressing numerically the intensity
of the signals for hybridization. Based on the numerals, the
relative intensities (%) of spots for the DNAs derived from the
type-strains were calculated by regarding the signal intensity of a
spot for the DNA from Arthrobacter sp. S34, FERM BP-6450, as 100
and used as an index for the DNA homology between the microorganism
and the type-strains. The results are in Table 1.
TABLE-US-00001 TABLE 1 Signal intensity of Strain of microorganism
hybridization Arthrobacter atrocyaneus, ATCC 13752 42.0
Arthrobacter aurescens, ATCC 13344 12.4 Arthrobacter citreus, ATCC
11624 36.2 Arthrobacter crystallpoietes, ATCC 15481 31.6
Arthrobacter globiformis, ATCC 8010 55.1 Arthrobacter nicotianae,
ATCC 15236 18.8 Arthrobacter oxydans, ATCC 14358 28.3 Arthrobacter
pascens, ATCC 13346 24.6 Arthrobacter protophormiae, ATCC 19271
29.3 Arthrobacter ramosus, ATCC 13727 98.6 Arthrobacter
ureafaciens, ATCC 7562 42.3 Arthrobacter viscous, ATCC 19584 0.0
Arthrobacter sp. S34, FERM BP-6450 100
As shown in Table 1, the signal intensity of hybridization for the
spot of DNA from Arthrobacter ramosus type strain, ATCC 13727, was
as high as 98.6%. The data revealed that Arthrobacter sp. S34, FERM
BP-6450, had the highest homology with Arthrobacter ramosus
type-strain, ATCC 13727, among the 12 type strains used in this
Example. The results in the above shows that Arthrobacter sp. S34,
FERM BP-6450, is a novel microorganism nearly related to
Arthrobacter ramosus type-strain, ATCC 13727.
EXAMPLE 2
Non-reducing Saccharide-forming Enzyme
Experiment 2-1
Preparation of Enzyme
A nutrient culture medium, consisting of 1.0 w/v % "PINE-DEX #4", a
dextrin commercialized by Matsutani Chemical Ind., Tokyo, Japan,
0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % monosodium
phosphate, 0.06 w/v % dipotassium hydrogen phosphate, 0.05 w/v %
magnesium sulfate, and water, was prepared and adjusted to pH 7.0.
About 100 ml aliquots of the medium were placed in 500-ml
Erlenmeyer flasks which were then autoclaved at 120.degree. C. for
20 min and cooled, followed by an inoculation of a seed of
Arthrobacter sp. S34, FERM BP-6450 and a culture at 37.degree. C.
for 48 hours under stirring conditions of 260 rpm for obtaining a
seed culture.
Except for containing 0.05 w/v % of "KM-75", a antifoamer
commercialized by Shin-Etsu Chemical, Co., Ltd, Tokyo, Japan, an
about 20 l of the same nutrient culture medium as used in the seed
culture was placed in a 30-l fermenter, sterilized, cooled to
37.degree. C., and inoculated with one v/v % of the seed culture to
the medium, followed by an incubation at 37.degree. C. and pHs of
5.5-7.5 for about 72 hours under aeration-agitation conditions.
A portion of the resultant culture was sampled, centrifuged to
separate into cells and a culture supernatant. The cells were
ultrasonically disrupted and centrifuged to collect supernatant for
a cell extract. Assay for non-reducing saccharide-forming enzyme
activity in each culture supernatant and cell extract revealed that
the former showed a relatively-low enzyme activity and the latter
exhibited an about 0.1 unit with respect to one milliliter of the
culture.
EXAMPLE 2-2
Purification of Enzyme
An about 80 l of a culture, obtained according to the method in
Example 2-1, was centrifuged at 8,000 rpm for 30 min to obtain an
about 800 g cells by wet weight. The wet cells were suspended in
two liters of 10 M phosphate buffer (pH 7.0) and treated with
"MODEL UH-600", an ultrasonic homogenizer commercialized by SMT
Co., Tokyo, Japan. The resulting solution was centrifuged at 10,000
rpm for 30 min to yield an about 2 l of a culture supernatant. To
and in the culture supernatant was added and dissolved ammonium
sulfate to give a saturation degree of 0.7, and the mixture was
allowed to stand at 4.degree. C. for 24 hours and centrifuged at
10,000 rpm for 30 min to obtain a precipitate. The precipitate thus
obtained was dissolved in 10 mM phosphate buffer (pH 7.0) and
dialyzed against a fresh preparation of the same buffer as above
for 48 hours, followed by centrifuging the dialyzed inner solution
at 10,000 rpm for 30 min to remove insoluble substances. An about
one liter of the resulting solution was subjected to an
ion-exchange column chromatography using a column packed with about
1.3 l of "SEPABEADS FP-DA13 GEL", an anion exchanger commercialized
by Mitsubishi Chemical Industries Ltd., Tokyo, Japan. The elution
step was carried out using a linear gradient buffer of 10 mM
phosphate buffer (pH 7.0) containing salt which increased from 0 M
to 0.6 M. The eluate from the column was fractionated, and the
fractions were respectively assayed for non-reducing
saccharide-forming enzyme activity. As a result, the enzyme
activity was remarkably found in fractions eluted with buffer
having a salt concentration of about 0.2 M, followed by pooling the
fractions.
Ammonium sulfate was added to the resulting solution to give a
concentration of 1 M, and the mixture was allowed to stand at
4.degree. C. for 12 hours, centrifuged at 10,000 rpm for 30 min to
collect a supernatant. The supernatant thus obtained was subjected
to hydrophobic column chromatography using a column packed with
"BUTYL TOYOPEARL 650M GEL", a hydrophobic gel commercialized by
Tosoh Corporation, Tokyo, Japan. The gel volume used was about 300
ml and used after equilibrated with 10 mM phosphate buffer (pH 7.0)
containing 1 M ammonium sulfate. The elution step was carried out
using a linear gradient buffer of 10 mM phosphate buffer (pH 7.0)
containing ammonium sulfate which decreased from 1 M to 0 M during
the feeding. The eluate from the column was fractionated, and the
fractions were respectively assayed for non-reducing
saccharide-forming enzyme activity. As a result, the enzyme
activity was remarkably found in fractions eluted with buffer
having a salt concentration of about 0.75 M, followed by pooling
the fractions.
The resulting solution was dialyzed against 10 mM phosphate buffer
(pH 7.0), and the resulting dialyzed inner solution was centrifuged
at 10,000 rpm for 30 min to collect a supernatant, followed by
subjecting the supernatant to ion-exchange column chromatography
using a column packed with about 40 ml of "DEAE TOYOPEARL 650S
GEL", an anion exchanger commercialized by Tosoh Corporation,
Tokyo, Japan. The elution step was carried out using a linear
aqueous salt solution which increased from 0 M to 0.2 M during the
feeding. The eluate from the column was fractionated, and the
fractions were respectively assayed for non-reducing
saccharide-forming enzyme activity. As a result, the enzyme
activity was remarkably found in fractions eluted with buffer
having a salt concentration of about 0.15 M, followed by pooling
the fractions. The resulting solution was further subjected to gel
filtration column chromatography using a column packed with about
380 ml of "ULTROGEL.RTM. AcA44 GEL", a gel for gel filtration
column chromatography commercialized by Sepracor/IBF s.a.
Villeneuve la Garenne, France, followed by collecting fractions
with the desired enzyme activity. The level of the non-reducing
saccharide-forming enzyme activity, specific activity, and yields
in the above purification steps are in Table 2.
TABLE-US-00002 TABLE 2 Enzyme activity of non-reducing Specific
activity Yield Purification step saccharide-forming enzyme (unit/mg
protein) (%) Cell extract 8,000 -- 100 Dialyzed inner-solution
7,500 0.2 94 after salting out with ammonium salt Eluate from
SEPABEADS column 5,200 0.7 65 Eluate from hydrophobic column 2,600
6.3 33 Eluate from TOYO PEARL 910 67.4 11 Eluate of gel filtration
59.0 168 0.7
The solution eluted and collected from the above gel filtration
chromatography was in a usual manner subjected to electrophoresis
using 7.5 w/v % polyacrylamide gel and resulted in a single protein
band. The data shows that the eluate from gel filtration
chromatography was a purified specimen of a non-reducing
saccharide-forming enzyme purified up to an electrophoretically
homogeneous form.
EXAMPLE 2-3
Property of Enzyme
EXAMPLE 2-3(a)
Action
A 20% aqueous solution containing glucose, maltose, maltotriose,
maltotetraose, maltopentaose, maltohexaose or maltoheptaose as a
substrate for enzyme was prepared, mixed with two units/g
substrate, d.s.b., of a purified specimen of a non-reducing
saccharide-forming enzyme obtained by the method in Example 2-2,
and enzymatically reacted at 50.degree. C. and pH 6.0 for 48 hours.
The reaction mixture was desalted and analyzed on high-performance
liquid chromatography (abbreviated as "HPLC" hereinafter) using two
columns of "MCI GEL CK04SS COLUMN", commercialized by Mitsubishi
Chemical Industries Ltd., Tokyo, Japan, which were cascaded in
series, followed by determining the saccharide composition of the
reaction mixture. The conditions and apparatus used in HPLC were as
follows: The column was kept at 85.degree. C. using "Co-8020", a
column oven commercialized by Tosoh Corporation, Tokyo, Japan.
Water as a moving phase was fed at a flow rate of 0.4 ml/min. The
eluate was analyzed on "RI-8020", a differential refractometer
commercialized by Tosoh Corporation, Tokyo, Japan. The results were
in Table 3.
TABLE-US-00003 TABLE 3 Elution time Percentage Substrate Reaction
product (min) (%) Glucose Glucose 57.2 100.0 Maltose Maltose 50.8
100.0 Maltotriose Glucosyltrehalose 43.2 36.2 Maltotriose 46.2 63.8
Maltotetraose Maltosyltrehalose 38.9 87.2 Maltotetraose 42.3 12.8
Maltopentaose Maltotriosyltrehalose 35.4 93.0 Maltopentaose 38.4
7.0 Maltohexaose Maltotetraosyltrehalose 32.7 93.8 Maltohexaose
35.2 6.2 Maltoheptaose Maltopentaosyltrehalose 30.2 94.2
Maltoheptaose 32.4 5.8
As evident form the results in Table 3, each reaction product
consisted essentially of the remaining substrate and a newly formed
non-reducing saccharide of .alpha.-glucosyltrehalose,
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, or .alpha.-maltopentaosyltrehalose
(in Table 3, it is expressed as glucosyltrehalose,
maltosyltrehalose, maltotriosyltrehalose, maltotetraosyltrehalose,
or maltopentaosyltrehalose). Substantially no other saccharide was
detected in the reaction mixture. Regarding and evaluating the
percentage of non-reducing saccharide in each reaction product as a
production yield, it was revealed that the yield of
.alpha.-glucosyltrehalose having a glucose polymerization degree of
3 was relatively low and the yield of those having a glucose
polymerization degree of 4 or higher such as
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose was as high as about 85% or higher.
No formation of non-reducing saccharide from glucose and maltose
was observed.
EXAMPLE 2-3(b)
Molecular Weight
A purified specimen of a non-reducing saccharide-forming enzyme,
obtained by the method in Example 2-2, was subjected to SDS-PAGE
using 10 w/v % polyacrylamide gel in a usual manner in parallel
with molecular markers commercialized by Japan Bio-Rad
Laboratories, Tokyo, Japan. Comparing with the positions of the
molecular markers after electrophoresis, the non-reducing
saccharide-forming enzyme exhibited a molecular weight of about
75,000.+-.10,000 daltons.
EXAMPLE 2-3(c)
Isoelectric Point
A purified specimen of a non-reducing saccharide-forming enzyme,
obtained by the method in Example 2-2, was isoelectrophoresed using
a polyacrylamide gel containing 2 w/v % "AMPHOLINE", an ampholyte,
commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden.
After isoelectrophoresis, the measurement of the pH of gel revealed
that the non-reducing saccharide-forming enzyme had an isoelectric
point of about 4.5.+-.0.5.
EXAMPLE 2-3(d)
Optimum Temperature and pH
Using a purified specimen of a non-reducing saccharide-forming
enzyme, obtained by the method in Example 2-2, it was examined the
influence of temperature and pH on the activity of the non-reducing
saccharide-forming enzyme. When examining the influence of
temperature, it was conducted similarly as in the assay for enzyme
activity except for reacting the enzyme at different temperatures.
In the examination of the influence of pH, it was conducted
similarly as in the assay for enzyme activity except for reacting
the enzyme at different pHs using appropriate 20 mM buffers. In
each examination, a relative value (%) of a lowered level of
reducing power of substrate in each reaction system was calculated
into its corresponding relative enzyme activity (%). FIG. 1 shows a
result of the influence of temperature, and FIG. 2 is of pH. The
cross axles in FIGS. 1 and 2 show reaction temperatures and
reaction pHs, respectively. As shown in FIG. 1, the optimum
temperature of the enzyme was about 50.degree. C. when incubated at
pH 6.0 for 60 min. Also as shown in FIG. 2, the optimum pH of the
enzyme was a pH of about 6.0 when incubated at 50.degree. C. for 60
min.
EXAMPLE 2-3(e)
Thermal and pH Stabilities
Using a purified specimen of a non-reducing saccharide-forming
enzyme, obtained by the method in Example 2-2, it was examined the
thermal and pH stabilities of the enzyme. The thermal stability was
examined by diluting the specimen with 20 mM phosphate buffer (pH
7.0), incubating the dilutions at prescribed temperatures for 60
min, cooling the incubated dilutions, and determining the remaining
enzyme activity in the dilutions according to the method of the
assay for the enzyme activity. The pH stability of the enzyme was
examined by diluting the specimen with 50 mM buffers with
appropriate different pHs, incubating the dilutions at 4.degree. C.
for 24 hours, adjusting the dilutions to pH 6, and determining the
remaining enzyme activity in the dilutions according to the method
of the assay for the enzyme activity. The results of the thermal
and pH stabilities of the enzyme are respectively shown in FIGS. 3
and 4. The cross axles in FIGS. 3 and 4 show incubation
temperatures and pHs for the enzyme, respectively. As shown in FIG.
3, the enzyme was stable up to about 55.degree. C. and was stable
at pHs in the range of about 5.0 to about 10.0 as shown in FIG.
4.
These results evidence that the non-reducing saccharide
forming-enzyme, obtained by the method in Example 2-2, is the
present non-reducing saccharide-forming enzyme having an optimum
temperature in a medium temperature range.
EXAMPLE 2-4
Partial Amino Acid Sequence
A portion of a purified specimen of a non-reducing
saccharide-forming enzyme, obtained by the method in Example 2-2,
was dialyzed against distilled water to obtain an about 80 .mu.g of
a sample by weight as a protein for analyzing the N-terminal amino
acid sequence. Using "PROTEIN SEQUENCER MODEL 473A", a protein
sequencer commercialized by Applied Biosystems, Inc., Foster City,
USA, the N-terminal amino acid sequence was analyzed up to 20 amino
acid residues from the N-terminus. The revealed N-terminal amino
acid sequence was the partial amino acid sequence of SEQ ID NO:4. A
portion of a purified specimen of a non-reducing saccharide-forming
enzyme, obtained by the method in Example 2-2, was dialyzed against
10 mM Tris-HCl buffer (pH 9.0) and in a usual manner concentrated
up to an about one mg/ml solution using "ULTRACENT-30", an
ultrafiltration membrane commercialized by Tosoh Corporation,
Tokyo, Japan. To 0.2 ml of the concentrate was added 10 .mu.g
"TPCK-TRYPSIN", a reagent trypsin commercialized by Wako Pure
Chemical Industries, Ltd., Tokyo, Japan, allowed to react at
30.degree. C. for 22 hours to digest the enzyme to form peptides.
The peptides were separated by subjecting the reaction mixture to
reverse-phase HPLC using ".mu. BONDASPHERE C18 COLUMN" having a
diameter of 3.9 mm and a length of 150 mm, a product of Waters
Chromatography Div., MILLIPORE Corp., Milford, USA. The elution
step was carried out at ambient temperature by feeding to the
column an aqueous solution containing 0.1 v/v % trifluoro acetate
and acetonitrile increasing from 24 to 48 v/v % for 60 min during
the feeding at a flow rate of 0.9 ml/min. The peptides eluted from
the column were detected by monitoring the absorbance at a
wavelength of 210 nm. Two peptides, which were well separated from
others, i.e., "S5" eluted at a retention time of about two hours
and "S8" eluted at a retention time of about 30 min were separated,
respectively dried in vacuo, and dissolved in 50 v/v % aqueous
acetonitrile solutions containing 50 .mu.l of 0.1 v/v % trifluoro
acetate. The peptide solutions were subjected to the protein
sequencer to analyze up to 20 amino acid residues. From peptides
"S5" and "S8" the amino acid sequences of SEQ ID Nos:5 and 6 were
obtained.
EXAMPLE 3
DNA Encoding Non-reducing Saccharide-forming Enzyme
EXAMPLE 3-1
Construction and Screening of Gene Library
Except for setting temperature and time for culture were
respectively set to 27.degree. C. and 24 hours, Arthrobacter sp.
S34, FERM BP-6450, was cultured similarly as in Example 2-1.
The culture was centrifuged to remove cells which were then
suspended in an adequate amount of Tris-EDTA-salt bufferred saline
(hereinafter designated as "TES buffer") (pH 8.0), admixed with
lysozyme in an amount of 0.05 w/v % to the cell suspension by
volume, followed by an incubation at 37.degree. C. for 30 min. The
resultant mixture was freezed by standing at -80.degree. C. for one
hour, and then admixed and sufficiently stirred with a mixture of
TES buffer and phenol preheated to 60.degree. C., cooled, and
centrifuged to collect the formed supernatant. To the supernatant
was added cold ethanol was added, and then the formed sediment was
collected, dissolved in an adequate amount of SSC buffer (pH 7.1),
admixed with 7.5 .mu.g ribonuclease and 125 .mu.g protease, and
incubated at 37.degree. C. for one hour. The resulting mixture was
admixed and stirred with chloroform/isoamyl alcohol, and allowed to
stand, followed by collecting the formed upper layer, adding cold
ethanol to the layer, and collecting the formed sediment. The
sediment was rinsed with a cold 70 v/v ethanol, dried in vacuo to
obtain a DNA, followed by dissolving in SSC buffer (pH 7.1) to give
a concentration of about one mg/ml, and freezing at -80.degree.
C.
Fifty microliters of the DNA was provided, admixed with an abut 50
units of KpnI as an restriction enzyme, and incubated at 37.degree.
C. for one hour to digest the DNA. Three micrograms of the digested
DNA and 0.3 microgram of "pBluescript II SK (+)", a plasmid vector
commercialized by Stratagene Cloning Systems, California, USA, was
weighed, subjected to the action , were ligated using "DNA LIGATION
KIT", commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan,
according to the protocol affixed to the kit. According to
conventional competent cell method, 100 .mu.l of "Epicurian Coli
XL1-Blue", an Escherichia coli strain commercialized by Stratagene
Cloning Systems, California, USA, was transformed with the ligated
product. Thus a gene library was obtained.
The gene library thus obtained was inoculated to a agar nutrient
plate medium (pH 7.0) containing 10 g/l tryptone, 5 g/l yeast
extract, 5 g/l sodium chloride, 75 mg ampicillin sodium salt, and
50 mg/l 5-bromo-4-chloro-indolyl-.beta.-galactoside, and incubated
at 37.degree. C. for 18 hours. About 5,000 white colonies formed on
the medium were in a usual manner fixed on "HYBOND-N-+", a nylon
film commercialized Amersham Corp., Div. Amersham International,
Arlington Heights, Ill., USA. Based on 1-8 amino acid residues in
the amino acid sequence of SEQ ID NO:5 revealed in Example 2-4, an
oligonucleotide having the nucleotide sequence of SEQ ID NO:18 was
chemically synthesized, and in a usual manner labelled with
[.gamma.-.sup.32P] ATP and T4 polynucleotide kinase to obtain a
probe. Using the probe, the colonies, which had been fixed on the
nylon film and obtained previously, were screened by conventional
colony hybridization method. The hybridization was carried out at
65.degree. C for 16 hours in a solution for hybridization
containing 6.times.SSC, 5.times. Denhalt solution, and 100 mg/l of
denatured salmon sperm DNA. The above nylon film after the
hybridization was washed with 6.times.SSC at 65.degree. C. for 30
min, and further washed with 2.times.SSC containing 0.1 w/v % SDS
at 65.degree. C. for two hours. The resulting nylon film was in a
usual manner subjected to autoradiography, and then, based on the
signals observed on the autoradiography, a colony which strongly
hybridized with the probe was selected and named "GY1" as a
transformant.
EXAMPLE 3-2
Decoding of Nucleotide Sequence
According to conventional manner, the transformant GY1 was
inoculated to L-broth (pH 7.0) containing 100 .mu.g/ml ampicillin
in a sodium form, and cultured at 37.degree. C. for 24 hours under
shaking conditions. After completion of the culture, the
proliferated cells were collected from the culture by
centrifugation and treated with conventional alkali-SDS method to
extract a recombinant DNA. The recombinant DNA was named pGY1.
Using the above probe, the recombinant DNA, pGY1, was analyzed on
conventional Southern blot technique, and based on the analytical
data a restriction map was constructed as shown in FIG. 5. As shown
in FIG. 5, it was revealed that the recombinant DNA, pGY1,
contained a nucleotide sequence consisting of bases of about 5,500
base-pairs (bp) from Arthrobacter sp. S34, FERM BP-6450, expressed
with a bold line, and that the recombinant DNA contained a
nucleotide sequence encoding the present non-reducing
saccharide-forming enzyme, as indicated with a black arrow within
the area of the bold line, in the area consisting bases of about
4,000 bp between two recognition sites by a restriction enzyme,
EcoRI. Based on the result, the recombinant DNA, pGY1, was
completely digested with EcoRI, and then a DNA fragment of about
4,000 bp was separated and purified using conventional agarose gel
electrophoresis. The DNA fragment and "pBluescript II SK (+)", a
plasmid vector commercialized by Stratagene Cloning Systems,
California, USA, which had been previously digested with EcoRI,
were ligated with conventional ligation method. With the ligated
product, "XL1-BLUE", an Escherichia coli strain commercialized by
Stratagene Cloning Systems, California, USA, was transformed to
obtain a transformant. A recombinant DNA was extracted from the
transformant in a usual manner, confirming in a usual manner that
it contained the aforesaid DNA fragment consisting of about bases
of 4,000 bp, and named it "pGY2". The transformant introduced with
"pGY2" was named "GY2".
The analysis of the nucleotide sequence of the recombinant DNA pGY2
on conventional dideoxy method revealed that it contained the
nucleotide sequence of SEQ ID NO:19 consisting bases of 3252 bp
derived from Arthrobacter sp. S34, FERM BP-6450. The nucleotide
sequence encodes the amino acid sequence as shown in parallel in
SEQ ID NO:19. Comparing the amino acid sequences of SEQ D NOs:4 to
6 as partial amino acid sequences of the present non-reducing
saccharide-forming enzyme confirmed in Example 2-4, the amino acid
sequences of SEQ ID Nos:4 to 6 were perfectly coincided with the
amino acids 2-21, 619-638, and 98-117 in SEQ ID NO:19. These data
indicate that the present non-reducing saccharide-forming enzyme
obtained in Example 2 consists of the amino acids 2-757 of SEQ ID
NO:19, or has the amino acid Sequence of SEQ ID NO:1, and that the
enzyme of Arthrobacter sp. S34, FERM BP-6450, is encoded by a
nucleotide sequence of bases 746-3013 of SEQ ID NO:19, or encoded
by the nucleotide sequence of SEQ ID NO:7. The structure of the
recombinant DNA pGY2 is in FIG. 6.
The above-identified amino acid sequence of the present
non-reducing saccharide-forming enzyme obtained by the method in
Example 2, and amino acid sequences of known enzymes having a
non-reducing saccharide-forming activity were compared using
"GENETYX-MAC, VER. 8", a commercially available computer program
commercialized by Software Development Co., Ltd., Tokyo, Japan,
according to the method by Lipman, David J. in Science, Vol. 227,
pp. 1,435-1,441 (1985) to calculate their homology (%). The enzymes
used as known enzymes were those from Arthrobacter sp. Q36 and
Rhizobium sp. M-11 disclosed in Japanese Patent Kokai No.
322,883/95; Sulfolobus acidocaldarius, ATCC 33909, disclosed in
Japanese Patent Kokai No. 84,586/96; and Sulfolobus solfataricus
KM1 disclosed in Sai-Kohyo No. WO 95/34642. As disclosed in the
above publications, the conventional enzymes have optimum
temperatures other than a medium temperature range. The information
of amino acid sequences of conventional enzymes is obtainable from
the GeneBank, a DNA database produced by the National Institutes of
Health (NIH), USA, under the accession numbers of D63343, D64128,
D78001 and D83245. The obtained homologies are in Table 4.
TABLE-US-00004 TABLE 4 Origin of enzyme for amino acid Homology on
amino sequence(*) comparison acid sequence Rhizobium sp. M-11
(D78001) 56.9% Arthrobacter sp. Q36 (D63343) 56.6% Sulfolobus
solfataricus KM1 (D64128) 33.2% Sulfolobus acidocaldarius, 31.4%
ATCC 33909 (D83245) *Numerals in parentheses are access numbers to
the GeneBank.
As shown in Table 4, the present non-reducing saccharide-forming
enzyme in Example 2 showed a highest amino acid homology of 56.9%
with the enzyme from Rhizobium sp. M-11 among conventional enzymes
with optimum temperatures out of a medium temperature range. The
data indicates that the present non-reducing saccharide-forming
enzyme generally comprises an amino acid sequence with a homology
of at least 57% with the amino acid sequence of SEQ ID NO:1. The
comparison result on amino acid sequence revealed that the enzyme
in Example 2 and the above-identified four types of conventional
enzymes have common amino acid sequences of SEQ ID NOs:2 and 3. The
enzyme in Example 2 has partial amino acid sequences of SEQ ID
NOs:2 and 3 as they correspond to amino acids 84-89 and 277-282 in
SEQ ID NO:1. The four types of enzymes used as references have the
above partial amino acid sequences which are positioned at their
corresponding parts. Based on the fact that any of the present
enzyme in Example 2 and the enzymes as references have a common
activity of forming non-reducing saccharides having a trehalose
structure as an end unit from reducing partial starch hydrolysates,
it was indicated that the partial amino acid sequences of SEQ ID
NOs:2 and 3 correlated to the expression of such an enzyme
activity. These results show that the present non-reducing
saccharide-forming enzyme can be characterized in that it comprises
the amino acid sequences of SEQ ID NOs:2 and 3, and has an optimum
temperature in a medium temperature range.
EXAMPLE 3-3
Transformant Introduced with DNA
Based on the 5'- and 3'-termini of the nucleotide sequence of SEQ
ID NO:7, an oligonucleotide of the nucleotide sequences of SEQ ID
NOs:20 and 21 were chemically synthesized in a usual manner. As
sense- and anti-sense-primers, 85 ng of each of the oligonucleotide
and 100 ng of the recombinant DNA pGY2 in Example 3-2 as a template
were mixed in a reaction tube, and the mixture was admixed with
1.25 units of "PYROBEST", a thermostable DNA polymerase specimen
commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, together
with 5 .mu.l of a buffer affixed with the specimen and 4 .mu.l of a
dNTP mixture. The resulting mixture was brought up to a volume of
50 .mu.l with sterilized distilled water to effect PCR. The
temperature for PCR was controlled in such a manner that the
mixture was treated with 25 cycles of successive incubations of
95.degree. C. for one minute, 98.degree. C. for 20 seconds,
70.degree. C. for 30 seconds, and 72.degree. C. for four minutes,
and finally incubated at 72.degree. C. for 10 min. A DNA as a PCR
product was collected in a usual manner to obtain an about 2,300 bp
DNA. The DNA thus obtained was admixed with "pKK223-3", a plasmid
vector commercialized by Pharmacia LKB Biotechnology AB, Uppsala,
Sweden, which had been previously cleaved with a restriction
enzyme, EcoRI, and blunted by "DNA BLUNTING KIT" commercialized by
Takara Shuzo Co., Ltd., Tokyo, Japan, and ligated by conventional
ligation method. Thereafter, the ligated product was treated in a
usual manner to obtain a recombinant DNA introduced with the above
DNA consisting of bases of about 2,300 bp. Decoding of the
recombinant DNA showed that it comprised a nucleotide sequence
which two nucleotide sequences of 5'-ATG-3' and 5'-TGA-3' were
respectively added to the 5'- and 3'-termini of the nucleo-tide
sequence of SEQ ID NO:7. The DNA was named "pGY3". The structure of
the recombinant DNA pGY3 was in FIG. 7.
The recombinant DNA pGY3 was in a usual manner introduced into an
Escherichia Coli LE 392 strain, ATCC 33572, which had been
competented in conventional manner, to obtain a transformant.
Conventional alkali-SDS method was applied for the transformant to
extract a DNA, and then the extracted DNA was confirmed to be pGY3
in a usual manner and named "GY3". Thus a transformant introduced
with a DNA encoding the present non-reducing saccharide-forming
enzyme.
EXAMPLE 3-4
Transformant Introduced with DNA
Based on a nucleotide sequence in the downstream of the 3'-terminus
of a promotor in "pKK223-3", a plasmid vector commercialized by
Pharmacia LKB Biotechnology AB, Uppsala, Sweden, oligonucleotide
having the nucleotide sequences of SEQ ID NOs:22 and 23 were
synthesized in conventional manner, and phosphorylated their
5'-termini using T4 polynucleotide kinase. The phosphorylated
oligonucleotide were annealed, ligated with "pKK223-3", a plasmid
vector commercialized by Pharmacia LKB Biotechnology AB, Uppsala,
Sweden, which had been previously cleaved with restriction enzymes
of EcoRI and PstI, by conventional ligation method. According to
conventional method, the ligated product was introduced into an
Escherichia coli strain which was then cultured and treated with
alkali-SDS method to extract a DNA. The DNA thus obtained had a
similar structure to a plasmid vector "pKK223-3", and had
recognition sites by restriction enzymes of EcoRI, XbaI, SpeI, and
PstI at the downstream of the promoter. The present inventors named
the DNA a plasmid vector "pKK4".
Similarly as in Example 3-3, PCR was done except for using
oligonucleotide with the nucleotide sequences of SEQ ID NOs:24 and
25, which had been chemically synthesized based on the 5'- and
3'-terminal partial nucleotide sequences of SEQ ID NO:7. A DNA as a
PCR product was collected in a usual manner to obtain an about
2,300 bp DNA. The DNA thus obtained was cleaved with restriction
enzymes, XbaI and SpeI, and the above plasmid vector pKK4, which
had been cleaved with XbaI and SpeI, were ligated by conventional
ligation method. Thereafter, the ligated product was treated in a
usual manner to obtain a recombinant DNA with the nucleotide
sequence of SEQ ID NO:7. The recombinant DNA was named "pKGY1".
Using overlap extension method, which two steps PCR were applied
for and reported by Horthon, Robert M. in Methods in Enzymology,
Vol. 217, pp. 270-279 (1993), a nucleotide sequence in the upper
part of the 5'-terminus of SEQ ID NO:7 in the above DNA pKGY1 was
modified. PCR as a first step PCR-A was done similarly as in
Example 3-3 except for using, as sense- and anti-sense-primers,
oligonucleotide of the nucleotide sequences of SEQ ID NOs:26 and
27, which had been chemically synthesized based on the nucleotide
sequence of plasmid vector pKK4; and as a template the above
recombinant DNA pKGY1. In parallel, PCR as a first step PCR-B was
done similarly as in Example 3-3 except for using, as sense- and
anti-sense-primers, oligonucleotide of the nucleotide sequences of
SEQ ID NOs:28 and 29, which had been respectively chemically
synthesized in a usual manner based on the nucleotide sequence of
SEQ ID NO:7; and as a template the above recombinant DNA pKGY1. A
DNA as a product of the first step PCR-A was collected in a usual
manner to obtain an about 390 bp DNA. A DNA as a product in the
first stp PCR-B was collected in conventional manner to obtain an
about 930 bp DNA.
PCR, as a second step PCR-A, was done similarly as in Example 3-3
except for using as a template a DNA mixture, i.e., a product of
the first PCR-A and the first step PCR-B; as a sense primer the
oligonucleotide sequence of the nucleotide sequence of SEQ ID
NO:26; and as an anti-sense primer the oligonucleotide of the
nucleotide sequence of SEQ ID NO:30, which had been chemically
synthesized in conventional manner based on the nucleotide sequence
of SEQ ID NO:7. The DNA as a product in the PCR was collected in a
usual manner to obtain an about 1,300 bp DNA.
The DNA as a product in the second PCR-A was cleaved with
restriction enzymes of EcoRI and BsiWI, and the formed DNA
consisting of bases of about 650 bp was collected in a usual
manner. An about 6,300 bp DNA, which was formed after cleavage of
the above recombinant DNA pKGY1 with restriction enzymes of EcoRI
and BsiWI, was collected in conventional manner. These DNAs were
ligated in a usual manner, and the ligated product was treated in
conventional manner to obtain a recombinant DNA comprising an about
650 bp DNA derived from the second step PCR-A. Decoding of the DNA
by conventional dideoxy method revealed that the obtained
recombinant DNA comprised a nucleotide sequence which the
nucleotide sequence of SEQ ID NO:8, a nucleotide sequence
represented by 5'-ATG-3', and a nucleotide sequence represented by
5'-TGA-3' were cascaded in the order as indicated above from the
5'-terminus to the 3'-terminus. The recombinant DNA thus obtained
was named "pGY4". The structure of pGY4 is substantially the same
as the recombinant DNA pGY3 except for that pGY4 comprises the
nucleotide sequence of SEQ ID NO:8.
The recombinant DNA pGY4 was introduced in conventional manner with
"BMH71-18mutS", an Escherichia coli competent cell commercialized
by Takara Shuzo Co., Ltd., Tokyo, Japan to obtain a transformant.
The transformant was treated with alkali-SDS method to extract a
DNA which was then identified with pGY4 in conventional manner.
Thus a transformant introduced with a DNA encoding the present
non-reducing saccharide-forming enzyme.
EXAMPLE 4
Preparation of Non-reducing Saccharide-Forming Enzyme
EXAMPLE 4-1
Preparation of Enzyme Using Microorganism of the Genus
Arthrobacter
In accordance with the method in Example 2-1, Arthrobacter sp. S34,
FERM BP-6450, was cultured by a fermenter for about 72 hours. After
the cultivation, the resulting culture was concentrated with an
SF-membrane to yield an about eight liters of a cell suspension.
The cell suspension was treated with "MINI-LABO", a supper
high-pressure cell disrupter commercialized by Dainippon
Pharmaceutical Co., Ltd., Tokyo, to disrupt the cells. The
resulting solution was centrifuged to obtain an about 8.5 l of a
supernatant. When measured for non-reducing saccharide-forming
activity in the supernatant, it showed an about 0.1 unit of the
enzyme activity with respect to one milliliter of the culture.
Ammonium sulfate was added to the supernatant to brought up to a
saturation degree of about 0.7 to salt out, and the sediment was
collected by centrifugation, dissolved in 10 mM phosphate buffer
(pH 7.0), and dialyzed against a fresh preparation of the same
buffer. Except for using an about 2 l of an ion-exchange resin, the
resulting dialyzed inner solution was fed to ion-exchange column
chromatography using "SEPABEADS FP-DA13 GEL", an anion exchanger
commercialized by Mitsubishi Chemical Industries Ltd., Tokyo,
Japan, as described in Example 2-2, to collect fractions with
non-reducing saccharide-forming enzyme. The fractions were pooled,
dialyzed against a fresh preparation of the same buffer but
containing 1 M ammonium sulfate, and the resulting dialyzed inner
solution was centrifuged to collect the formed supernatant. Except
for using an about 300 ml gel, the supernatant was fed to
hydrophobic column chromatography in accordance with the method
described in Example 2-2 to collect fractions with non-reducing
saccharide-forming enzyme. Then it was confirmed that the obtained
enzyme had an optimum temperature over 40.degree. C. but below
60.degree. C., i.e., a temperature in a medium temperature range,
and an acid pH range of less than 7.
Thus an about 2,600 units of the present non-reducing
saccharide-forming enzyme was obtained.
EXAMPLE 4-2
Preparation of Enzyme Using Transformant
One hundred ml of an aqueous solution containing 16 g/l
polypeptone, 10 g/l yeast extract, and 5 g/l sodium chloride was
placed in a 500-ml Erlenmeyer flask, autoclaved at 121.degree. C.
for 15 min, cooled, adjusted aseptically to pH 7.0, and admixed
aseptically with 10 mg of ampicillin in a sodium salt to obtain a
liquid nutrient medium. The nutrient medium was inoculated with the
transformant GY2 in Example 3-2, and incubated at 37.degree. C. for
about 20 hours under aeration-agitation conditions to obtain a seed
culture. Seven liters of a medium having the same composition as
used in the seed culture was prepared as in the case of the seed
culture and placed in a 10-l fermenter, and inoculated with 70 ml
of the seed culture, followed by the incubation for about 20 hours
under aeration-agitation conditions. From the resultant culture
cells were collected by centrifugation in a usual manner. The
collected cells were suspended in phosphate buffer (pH 7.0),
disrupted by the treatment of ultrasonication, and centrifuged to
remove insoluble substances, followed by collecting a supernatant
to obtain a cell extract. The extract was dialyzed against 10 mM
phosphate buffer (pH 7.0). The resulting dialyzed inner solution
was collected and confirmed that it exhibited a non-reducing
saccharide-forming enzyme activity, had an optimum temperature in a
medium temperature range, i.e., a temperature of over 40.degree. C.
but below 60.degree. C., and had an optimum pH in an acid pH range,
i.e., a pH of less than 7.
Thus the present non-reducing saccharide-forming enzyme was
obtained. In the culture of this example, an about 0.2 unit/ml
culture of the enzyme was produced.
As a control, "XL1-BLUE", an Escherichia coli strain commercialized
by Stratagene Cloning Systems, California, USA, was cultured under
the same conditions as above in a nutrient culture medium of the
same composition as used in the above except that it contained no
ampicillin. Similarly as above, a cell extract was obtained and
dialyzed. No activity of non-reducing saccharide-forming enzyme was
detected in the resulting dialyzed inner solution, meaning that the
transformant GY2 is useful in producing the present non-reducing
saccharide-forming enzyme.
EXAMPLE 4-3
Preparation of Enzyme Using Transformant
The transformant GY3 in Example 3-3 was cultured similarly as in
Example 4-2 except for using a liquid nutrient culture medium
consisting of one w/v % maltose, three w/v polypeptone, one w/v %
"MEAST PIG", a product of Asahi Breweries, Ltd., Tokyo, Japan, 0.1
w/v % dipotassium hydrogen phosphate, 100 .mu.g/ml ampicillin, and
water. The resultant culture was treated with ultrasonication to
disrupt cells, and the resulting mixture was centrifuged to remove
insoluble substances. When assayed for non-reducing
saccharide-forming enzyme activity in the resulting supernatant,
the culture contained about 15 units/ml culture of the enzyme. In
accordance with the method in Example 2-2, the enzyme in the
supernatant was purified, confirming that the resulting purified
specimen exhibited a non-reducing saccharide-forming enzyme
activity, had an optimum temperature in a medium temperature range,
i.e., a temperature of over 40.degree. C. but below 60.degree. C.,
and had an optimum pH in an acid pH range, i.e., a pH of less than
7. Thus the present non-reducing saccharide-forming enzyme was
obtained.
EXAMPLE 4-4
Preparation of Enzyme Using Transformant
The transformant GY4 in Example 3-4 was cultured similarly as in
Example 4-2 except for using a liquid nutrient culture medium
consisting of two w/v % maltose, four w/v % peptone, one w/v %
yeast extract, 0.1 w/v % sodium dihydrogen phosphate, 200 .mu.g/ml
ampicillin, and water. The resultant culture was treated with
ultrasonication to disrupt cells, and the resulting mixture was
centrifuged to remove insoluble substances. When assayed for
non-reducing saccharide-forming enzyme activity in the resulting
supernatant, the culture contained about 60 units/ml culture of the
enzyme. In accordance with the method in Example 2-2, the enzyme in
the supernatant was purified, confirming that the resulting
purified specimen exhibited a non-reducing saccharide-forming
enzyme activity, had an optimum temperature in a medium temperature
range, i.e., a temperature of over 40.degree. C. but below
60.degree. C., and had an optimum pH in an acid pH range, i.e., a
pH of less than 7. Thus the present non-reducing saccharide-forming
enzyme was obtained.
EXAMPLE 5
Trehalose-releasing Enzyme
EXAMPLE 5-1
Production of Enzyme
According to the method in Example 2-1, Arthrobacter sp. S34, FERM
BP-6450, was cultured by a fermenter. Then, in accordance with the
method in Example 2-2, the resulting culture was sampled, followed
by separating the sample into cells and a supernatant. From the
cells a cell extract was obtained. When assayed for
trehalose-releasing activity of the supernatant and the cell
extract, the former scarcely exhibited the enzyme activity, while
the latter exhibited an about 0.3 uni/ml culture of the enzyme.
EXAMPLE 5-2
Preparation of Enzyme
An about 80 l of a culture, prepared according to the method in
Example 2-1, was centrifuged at 8,000 rpm for 30 min to obtain an
about 800 g cells by wet weight. Two l of the wet cells was
suspended in 10 mM phosphate buffer (pH 7.0) and treated with
"MODEL UH-600", an ultrasonic homogenizer commercialized by MST
Co., Tokyo, Japan. The resulting suspension was centrifuged at
10,000 rpm for 30 min, followed a collection of an about two liters
of a supernatant. The supernatant was admixed with ammonium sulfate
to bring to a saturation degree of 0.7, allowed to stand at
4.degree. C. for 24 hours, and centrifuged at 10,000 rpm for 30 min
to obtain a precipitate salted out with ammonium sulfate. The
precipitate was dissolved in 10 mM phosphate buffer (pH 7.0),
dialyzed against a fresh preparation of the same buffer for 48
hours, and centrifuged at 10,000 rpm for 30 min to remove insoluble
substances. An about one liter of the resulting dialyzed inner
solution was fed to ion-exchange column chromatography using an
about 1.3 l of "SEPABEADS FP-DA13 GEL", an anion exchanger
commercialized by Mitsubishi Chemical Industries Ltd., Tokyo,
Japan. The elution step was carried out using a linear 10 mM
phosphate buffer (pH 7.0) containing salt decreasing from 0 M to
0.6 M during the feeding. The eluate from the column was
fractionated, and the fractions each were assayed for
trehalose-releasing enzyme activity. As a result, the enzyme
activity was remarkably found in fractions eluted with buffer
having a salt concentration of about 0.2 M, followed by pooling the
fractions.
Ammonium sulfate was added to the pooled solution to bring to a
concentration of 1 M, and the mixture was allowed to stand at
4.degree. C. for 12 hours, centrifuged at 10,000 rpm for 30 min to
collect a supernatant. The supernatant was subjected to hydrophobic
column chromatography using a column packed with "BUTYL TOYOPEARL
650M GEL", a hydrophobic gel commercialized by Tosoh Corporation,
Tokyo, Japan. Prior to use, the gel volume was set to about 300 ml
and equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1
M ammonium sulfate. The elution step was carried out using a linear
gradient aqueous solution of ammonium decreasing from 1 M to 0 M
during the feeding. The eluate from the column was fractionated,
and the fractions were respectively assayed for trehalose-releasing
enzyme activity. As a result, the enzyme activity was remarkably
found in fractions eluted with buffer having an ammonium
concentration of about 0.5 M, followed by pooling the
fractions.
The fractions were pooled, dialyzed against 10 mM phosphate buffer
(pH 7.0), and the dialyzed inner solution was centrifuged at 10,000
rpm for 30 min. Then the resulting supernatant was collected and
subjected to "DEAE-TOYOPEARL 650S GEL", an anion exchanger
commercialized by Tosoh Corporation, Tokyo, Japan. The elution step
was carried out using a linear gradient aqueous solution of salt
increasing from 0 M to 0.2 M during the feeding. The eluate from
the column was fractionated, and the fractions were respectively
assayed for trehalose-releasing enzyme activity. As a result, the
enzyme activity was remarkably found in fractions eluted with
buffer having an ammonium concentration of about 0.15 M, followed
by pooling the fractions. The pooled solution was subjected to gel
filtration chromatography using about 380 ml of "ULTROGEL.RTM.
AcA44 RESIN", a gel for gel filtration column chromatography
commercialized by Sepracor/IBF s.a. Villeneuve la Garenne, France,
followed collecting fractions with a remarkable activity of the
enzyme. The content, specific activity, and yield of the enzyme in
each step are in Table 5.
TABLE-US-00005 TABLE 5 Activity of Trehalose- Specific releasing
activity Yield Step enzyme (unit) (mg/protein) (%) Cell extract
24,000 -- 100 Dialyzed inner solution 22,500 0.6 94 after salting
out with ammonium sulfate Eluate from SEPABEADS column 15,600 2.0
65 Eluate from hydrophobic column 6,400 25.3 27 Eluate from
TOYOPEARL column 4,000 131 17 Eluate after gel filtration 246 713
1.0
When electrophoresed in 7.5 w/v % polyacrylamide gel in
conventional manner, the solution eluted and collected from the
above gel filtration chromatography gave a single protein band. The
data indicates that the eluate from gel filtration chromatography
obtained in the above was a purified trehalose-releasing enzyme
purified up to an electrophoretically homogeneous level.
EXAMPLE 5-3
Property of Enzyme
EXAMPLE 5-3(a)
Action
Any one of saccharides consisting of .alpha.-glucosyltrehalose,
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose as non-reducing saccharides having
a trehalose structure obtained by the method in the later described
Example 8-3; and maltotriose, maltotetraose, maltopentaose,
maltohexaose, and maltoheptaose as reducing saccharides was
dissolved in water into a 2 w/v % solution as an aqueous substrate
solution for substrate. Each aqueous substrate solution was admixed
with two units/g substrate, d.s.b., of a purified specimen of
trehalose-releasing enzyme obtained by the method in Example 5-2,
and enzymatically reacted at 50.degree. C. and pH 6.0 for 48 hours.
In accordance with the method in Example 2-3(a), the reaction
product was analyzed on HPLC after desalting to calculate the
saccharide composition of the reaction products each. The results
are in Table 6. In Table 6, .alpha.-glucosyltrehalose,
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose were respectively expressed as
glucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose,
maltotetraosyltrehalose, and maltopentaosyltrehalose.
TABLE-US-00006 TABLE 6 Elution Composi- time tion Substrate
Reaction product (min) (%) Glucosyltrehalose Trehalose 48.5 16.8
Glucose 57.2 8.2 Glucosyltrehalose 43.3 75.0 Maltosyltrehalose
Trehalose 48.5 44.1 Maltose 50.8 44.4 Maltosyltrehalose 38.9 11.5
Maltotriosyltrehalose Trehalose 48.5 40.5 Maltotriose 46.2 59.0
Maltotriosyltrehalose 35.4 0.5 Maltotetraosyltrehalose Trehalose
48.5 35.0 Maltotetraose 42.1 64.2 Maltotetraosyltrehalose 32.7 0.3
Maltopentaosyltrehalose Trehalose 48.5 29.5 Maltopentaose 38.2 70.2
Maltopentaosyltrehalose 30.2 0.3 Maltotriose Maltotriose 46.2 100.0
Maltotetraose Maltotetraose 42.1 100.0 Maltopentaose Maltopentaose
38.2 100.0 Maltohexaose Maltohexaose 35.2 100.0 Maltoheptaose
Maltoheptaose 32.6 100.0
As evident from the results in Table 6, the trehalose-releasing
enzyme, obtained by the method in Example 5-2, specifically
hydrolyzed a non-reducing saccharide, which has a trehalose
structure as an end unit and a glucose polymerization degree of at
least three, to release trehalose from the rest of the non-reducing
saccharide to form trehalose and a reducing saccharide having a
glucose polymerization degree of one or more. While the enzyme did
not act on maltooligosaccharides such as maltotriose and lower
saccharides.
EXAMPLE 5-3(b)
Molecular Weight
A purified specimen of a trehalose-releasing enzyme, obtained by
the method in Example 5-2, was subjected along with molecular
markers commercialized by Japan Bio-Rad Laboratories, Tokyo, Japan,
to conventional SDS-PAGE using 10 w/v % polyacrylamide gel. After
electrophoresis, the position of the specimen electrophoresed on
the gel was compared with those of the markers, revealing that the
specimen had a molecular weight of about 62,000.+-.5,000
daltons.
EXAMPLE 5-3(c)
Isoelectric Point
A purified specimen of a trehalose-releasing enzyme, obtained by
the method in Example 5-2, was in a usual manner subjected to
isoelectrophoresis using a polyacrylamide gel containing 2 w/v %
"AMPHOLINE", an ampholyte commercialized by Pharmacia LKB
Biotechnology AB, Uppsala, Sweden. Measurement of pH of the gel
after electrophoresis, it had an isoelectric point of about
4.7.+-.0.5.
EXAMPLE 5-3(d)
Optimum Temperature and pH
A purified specimen of a trehalose-releasing enzyme, obtained by
the method in Example 5-2, was examined on the influence of the
temperature and pH on the enzyme activity. The influence of
temperature was examined according to the assay for enzyme activity
except for reacting the enzyme at different temperatures. The
influence of pH was examined according to the assay for enzyme
activity except for reacting the enzyme at different pHs using
appropriate 20 mM buffers. In each procedure, relative values (%)
of the increased level of reducing power found in each system were
calculated and regarded as relative enzyme activity (%). The
results of the influence of temperature and pH are respectively in
FIGS. 8 and 9. The cross axles in FIGS. 8 and 9 show reaction
temperatures and pHs for the enzyme, respectively. As shown in FIG.
8, the optimum temperature of the enzyme was about 50 to about
55.degree. C. when incubated at pH 6.0 for 30 min, while the
optimum pH of the enzyme was a pH of about 6.0 when incubated at
50.degree. C. for 30 min.
EXAMPLE 5-3(e)
Stability on Temperature and pH
A purified specimen of a trehalose-releasing enzyme, obtained by
the method in Example 5-2, was examined on the stability of
temperature and pH. The stability of temperature was examined by
diluting the specimen with 20 mM phosphate buffer (pH 7.0),
incubating the dilutions at different temperatures for 60 min,
cooling the resulting dilutions, and assaying the enzyme activity
remained in the dilutions. The pH stability was studied by diluting
the specimen with 50 mM buffers (pH 7.0) with different pHs,
incubating the dilutions at 4.degree. C. for 24 hours, adjusted to
pH 6, and assaying the enzyme activity remained in the dilutions.
The results of the stability of temperature and pH are respectively
in FIGS. 10 and 11. The cross axles in FIGS. 10 and 11 show
temperatures and pHs at which the enzyme was kept, respectively. As
shown in FIG. 10, the enzyme was stable up to about 50.degree. C.,
while the enzyme was stable at pHs in the range of about 4.5 to
about 10.0.
The results described hereinbefore indicate that the
trehalose-releasing enzyme, obtained by the method in Example 5-2,
is the present enzyme which has an optimum temperature in a medium
temperature range.
EXAMPLE 5-4
Partial Amino Acid Sequence
A portion of a purified specimen of a trehalose-releasing enzyme,
obtained by the method in Example 5-2, was dialyzed against
distilled water and prepared into a sample containing about 80 ng
protein for the N-terminal amino acid analysis. Using "PROTEIN
SEQUENCER MODEL 473A", a protein sequencer commercialized by
Applied Biosystems, Inc., Foster City, USA, the N-terminal amino
acid sequence was analyzed up to 20 amino acid residues from the
N-terminus. The revealed N-terminal amino acid sequence was the
partial amino acid sequence of SEQ ID NO:14.
A portion of a purified specimen of a trehalose-releasing enzyme,
obtained by the method in Example 5-2, was dialyzed against 10 mM
Tris-HCl buffer (pH 9.0) and in a usual manner concentrated to give
a concentration of about one milligram per milliliter using
"ULTRACENT-30", an ultrafiltration membrane commercialized by Tosoh
Corporation, Tokyo, Japan. To 0.2 ml of the concentrate was added
10 .mu.g of a lysyl endopeptidase reagent commercialized by Wako
Pure Chemical Industries, Ltd., Tokyo, Japan, and the mixture was
incubated at 30.degree. C. for 22 hours to digest the enzyme and to
form peptides. The reaction mixture was subjected to reverse-phase
HPLC using a column of "NOVA-PAK C18 COLUMN", 4.5 mm in diameter
and 150 mm in length, commercialized by Waters Chromatography Div.,
Millipore Corp., Milford, Mass., USA, to separate the peptides
under ambient temperature. The elution step was carried out using a
linear gradient of a 0.1 v/v % aqueous trifluoroacetic acid
solution containing acetonitrile increasing from 24 v/v % to 48 v/v
% for 60 min during the feeding at a flow rate of 0.9 ml/min.
Peptides eluate from the column was monitored by measuring at a
wavelength of 210 nm. Two peptides, named "RT18" with a retention
time of about 18 min and "RT33" with a retention time of about 33
min and well separated from others, were collected, dried in vacuo,
and dissolved respectively in a 50 v/v % aqueous acetonitrile
solution containing 200 .mu.l of 0.1 v/v % trifluoroacetic acid.
The peptide solutions were subjected to a protein sequencer to
analyze up to 20 amino acid residues from the N-terminus of each
peptide. The amino acid sequences of SEQ ID NOs:15 and 16 from the
peptides RT18 and RT33, respectively.
EXAMPLE 6
DNA Encoding Trehalose-releasing Enzyme
EXAMPLE 6-1
Construction and Screening of Gene Library
According to Example 3-1, a gene library of Arthrobacter sp. S34,
FERM BP-6450 was constructed, and then subjected to screening by
applying colony hybridization method under the conditions as used
in Example 3-1 except for using as a probe an oligonucleotide,
having a nucleotide sequence encoding the present
trehalose-releasing enzyme, prepared by the following procedures;
The probe was in a usual manner prepared by labelling with an
isotope of [.gamma.-.sup.32P] ATP and T4 polynucleotide kinase the
oligonucleotide having the nucleotide sequence of SEQ ID NO:31,
which had been chemically synthesized based on an amino acid
sequence consisting of amino acids 12-20 of SEQ ID NO:15 revealed
in Example 5-4. A transformant which strongly hybridized with the
prove was selected.
According to the method in Example 3-2, a recombinant DNA was
extracted from the transformant and analyzed on conventional
Southern blot technique using the above prove. A restriction map
made based on the analytical data was coincided with that of the
recombinant DNA pGY1 obtained in Examples 3-1 and 3-2. As shown in
FIG. 5, it was revealed that the present recombinant DNA in this
example contained a nucleotide sequence, which encoded the present
trehalose-releasing enzyme as indicated with an oblique arrow,
within a region consisting of bases of about 2,200 bp positioned
between recognition sites by restriction enzymes, PstI and KpnI.
Using the recombinant DNA pGY1, it was proceeded the decoding of
the nucleotide sequence of DNA encoding the present
trehalose-releasing enzyme.
EXAMPLE 6-2
Decoding of Nucleotide Sequence
The recombinant DNA pGY1, obtained by the method in Example 3-2,
was in conventional manner completely digested with a restriction
enzyme, PstI. The DNA fragment of about 3,300 bp formed in the
resulting mixture was removed on conventional agarose
electrophoresis, and the formed DNA fragment of about 5,200 bp was
collected. The DNA fragment was in a usual manner subjected to
ligation reaction, and the ligated product was used to transform
"XL1-BLUE", an Escherichia coli strain commercialized by Stratagene
Cloning Systems, California, USA. From the resultant transformant,
a recombinant DNA was extracted by conventional method. The
recombinant DNA was confirmed to have a region consisting of bases
of about 2,200 bp comprising a nucleotide sequence encoding the
present trehalose-releasing enzyme, and named "pGZ2". A
transformant intr254oduce with pGZ2 was named a recombinant DNA
pGZ2.
Analysis of Conventional dideoxy method for the nucleotide sequence
of the recombinant DNA pGZ2 revealed that it contained a nucleotide
sequence consisting of 2,218 bp bases as shown in SEQ ID NO:32
derived from Arthrobacter sp. S34, FERM BP-6450. The nucleotide
sequence could encode the amino acid sequence in SEQ ID NO:32. The
amino acid sequence was compared with those of SEQ ID NOs:14 to 16
as partial amino acid sequences of the present trehalose-releasing
enzyme confirmed in Example 5-4. As a result, the amino acid
sequences of SEQ ID NOs:14, 15 and 16 were respectively coincided
with amino acids 1-20, 298-317, and 31-50 of the amino acid
sequence in SEQ ID NO:32. The data indicates that the
trehalose-releasing enzyme in Example 5 comprises the amino acid
sequence in SEQ ID NO:32 or the one of SEQ ID NO:9, and that the
enzyme from Arthrobacter sp. S34, FERM BP-6450, is encoded by bases
477-2,201 in SEQ ID NO:32 or the nucleotide sequence of SEQ ID
NO:17. FIG. 12 shows the structure of the aforesaid recombinant DNA
pGZ2.
The above amino acid sequence of the present trehalose-releasing
enzyme, obtained by the method in Example 5, and other conventional
ones of enzymes having an activity of trehalose-releasing enzyme
were compared with each other in accordance with the method in
Example 3-2 to determine their homology (%). As conventional
enzymes, those derived from Arthrobacter sp. Q36 disclosed in
Japanese Patent Kokai No. 298,880/95; Rhizoblum sp. M-11, disclosed
in Japanese Patent Kokai No. 298,880/95; Sulfolobus acidocaldarius,
ATCC 33909; and Sulfolobus solfataricus KM1 disclosed in Sai-Kohyo
No. WO95/34642. All of these enzymes have optimum temperatures out
of a medium temperature range. The amino acid sequences of these
enzymes are available from the GenBank, a DNA database produced by
the National Institutes of Health (NIH), USA, under the accession
numbers of D63343, D64130, D78001, and D83245. The information of
their homology are in Table 7.
TABLE-US-00007 TABLE 7 Origin of enzyme for amino acid Homology on
amino sequence(*) comparison acid sequence Arthrobacter sp. Q36
(D63343) 59.9% Rhizobium sp. M-11 (D78001) 59.1% Sulfolobus
solfataricus KM1 (D64130) 37.7% Sulfolobus acidocaldarius, 36.0%
ATCC 33909 (D83245) *Numerals in parentheses are access numbers to
the GeneBank.
As shown in Table 7, the present trehalose-releasing enzyme in
Example 5 showed a highest amino acid homology of 59.9% with the
enzyme from Arthrobacter sp. Q36 among conventional enzymes with
optimum temperatures out of a medium temperature range. The data
indicates that the present trehalose-releasing enzyme generally
comprises an amino acid sequence with a homology of at least 60%
with the amino acid sequence of SEQ ID NO:9. The comparison result
on amino acid sequence revealed that the enzyme in Example 5 and
the above-identified four types of conventional enzymes have common
amino acid sequences of SEQ ID NOs:10 and 13. The enzyme in Example
5 has partial amino acid sequences of SEQ ID NOs:10 to 13 as found
in amino acids 148-153, 185-190, 248-254 and 285-291 in SEQ ID
NO:9. The four types of enzymes used as references have the above
partial amino acid sequences which are positioned at their
corresponding parts. Based on the fact that any of the present
enzyme in Example 5 and the enzymes as references have commonly an
activity of specifically hydrolysing a non-reducing saccharide,
which has a trehalose structure as an end unit and a glucose
polymerization degree of at least three, to release trehalose from
the rest of the non-reducing saccharide, it was indicated that the
partial amino acid sequences of SEQ ID NOs:10 to 13 correlated to
the expression of such an enzyme activity. These results show that
the present trehalose-releasing enzyme can be characterized in that
it comprises the amino acid sequences of SEQ ID NOs:10 to 13 and
has an optimum temperature in a medium temperature range.
EXAMPLE 6-3
Transformant Introduced with DNA
Based on the 5'- and 3'-terminal nucleotide sequences of SEQ ID
NO:17, oligonucleotides of the bases of SEQ ID NOs:33 and 34 were
chemically synthesized in a usual manner. As sense-and
anti-sense-primers, 85 ng of each of the oligonucleotides and 100
ng of the recombinant DNA pGZ2 in Example 6-2 as a template were
mixed in a reaction tube while adding another reagents in
accordance with Example 3-3. The temperature for PCR was controlled
in such a manner that the mixture was treated with 25 cycles of
successive incubations of 95.degree. C. for one minute, 98.degree.
C. for 20 seconds, 70.degree. C. for 30 seconds, and 72.degree. C.
for four minutes, and finally incubated at 72.degree. C. for 10
min. A DNA as a PCR product was collected in a usual manner to
obtain an about 1,700 bp DNA. The DNA thus obtained was admixed
with "pKK233-3", a plasmid vector commercialized by Pharmacia LKB
Biotechnology AB, Uppsala, Sweden, which had been previously
cleaved with a restriction enzyme, EcoRI, and blunted by "DNA
BLUNTING KIT" commercialized by Takara Shuzo Co., Ltd., Tokyo,
Japan, and ligated by conventional ligation method. Thereafter, the
ligated product was treated in a usual manner to obtain a
recombinant DNA introduced with the above DNA consisting of bases
of about 1,700 bp. Decoding of the recombinant DNA by conventional
dideoxy method showed that it comprised a nucleotide sequence which
a nucleotide sequence of 5'-TGA-3' was added to 3'-terminus of the
nucleotide sequence of SEQ ID NO:17. The DNA was named "pGZ3". The
structure of the recombinant DNA pGZ3 was in FIG. 13.
The recombinant pGZ3 was in a usual manner introduced into an
Escherichia coli LE 392 strain, ATCC 33572, which had been
competented in conventional manner, to obtain a transformant.
Conventional alkali-SDS method was applied for the transformant to
extract a DNA and named "GZ3" by identifying transformant as pGZ3.
Thus a transformant, introduced with the present
trehalose-releasing enzyme, was obtained.
EXAMPLE 6-4
Transformant Introduced with DNA
PCR was done similarly as in Example 6-3 except for using, as
sense- and anti-sense-primers, oligonucleotide having nucleotide
sequences of SEQ ID NOs:35 and 36, respectively, which had been
chemically synthesized based on the 5'- and 3'-terminal nucleotide
sequences of SEQ ID NO:17. A DNA as a PCR product was collected in
a usual manner to obtain an about 1,700 bp DNA. The DNA thus
obtained was cleaved with restriction enzymes, XbaI and SpeI, and
"pKK4", a plasmid vector obtained by the method in Example 3-4,
which had been previously cleaved with restriction enzyme, XbaI and
SpeI, were ligated in a usual manner. Thereafter, the ligated
product was treated in a usual manner to obtain a recombinant DNA
comprising the nucleotide sequence of SEQ ID NO:17. The recombinant
DNA thus obtained was named "pKGZ1".
A nucleotide sequence in the upper part of the 5'-terminus of SEQ
ID NO:17 contained in the recombinant DNA pKGZ1 was modified
similarly as in Example 3-4; PCR as a first PCR-C was carried out
similarly as in Example 3-3 except for using the above recombinant
DNA pKGZ1 as a template and oligonucleotides of SEQ ID NOs:26 and
37, as sense- and anti-sense-primers, which had been chemically
synthesized in a usual manner based on the nucleotide sequence of
the plasmid vector pKK4. In parallel, PCR as a first PCR-D was
carried out similarly as in Example 3-3 except for using the above
recombinant DNA pKGZ1 as a template and oligonucleotides of SEQ ID
NOs:38 and 39, as sense- and anti-sense-primers, which had been
chemically synthesized in a usual manner based on the nucleotide
sequences of SEQ ID NOs:38 and 39. A DNA as a PCR-C product was
collected in a usual manner to obtain an about 390 bp DNA, while
another DNA as a PCR-D product was collected similarly as above to
obtain an about 590 bp DNA.
PCR as a second PCR-B was carried out similarly as in Example 3-3
except for using the DNA mixture obtained as products in the first
PCR-C and first PCR-D, an oligonucleotide of SEQ ID NO:26 used in
the first PCP-C as a sense primer, and an oligonucleotide of SEQ ID
NO:39 used in the first PCR-D as an anti-sense primer. A DNA as a
PCR product was collected in a usual manner to obtain an about 950
bp DNA.
The DNA as a second PCR-B product was cleaved with a restriction
enzyme, EcoRI, and the formed about 270 bp DNA was collected in
conventional manner. The recombinant DNA pKGZ1 was cleaved with a
restriction enzyme, EcoRI, and the formed about 5,100 bp DNA was
collected similarly as above. These DNAs were ligated as usual and
treated in a usual manner to obtain a recombinant DNA comprising
about 270 bp DNA from the second PCR-B product. Decoding of the
recombinant DNA by conventional dideoxy method revealed that it
contained the nucleotide sequence of SEQ ID NO:8, one of SEQ ID
NO:17, and one represented by 5'-TGA-3' in the order as indicated
from the 5'- to 3'-termini. The recombinant DNA thus obtained was
named "pGZ4". The recombinant DNA pGZ4 had substantially the same
structure as the recombinant DNA pGZ3 obtained in Example 6-3
except that it had the nucleotide sequence of SEQ ID NO:8.
The recombinant DNA pGZ4 was introduced into "BMH71-18mutS", an
Escherichia coli competent cell commercialized by Takara Shuzo Co.,
Ltd., Tokyo, Japan, to obtain a transformant. Using conventional
alkali-SDS method, a DNA was extracted from the transformant and
confirmed to be pGZ4 according to conventional manner. It was named
"GZ4". Thus a transformant introduced with a DNA encoding the
present trehalose-releasing enzyme.
EXAMPLE 7
Preparation of Trehalose-Releasing Enzyme
EXAMPLE 7-1
Preparation of Enzyme Using Microorganisms of the Genus
Arthrobacter
A seed culture of Arthrobacter sp. S34, FERM BP-6450, was
inoculated to a nutrient culture medium and incubated by a
fermenter for about 72 hours in accordance with the method in
Example 2-1. After the incubation, the resultant culture was
filtered and concentrated with an SF-membrane to obtain an about
eight liters of cell suspension which was then treated with
"MINI-LABO", a super high-pressure cell disrupter commercialized by
Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, to disrupt cells.
The cell disruptant was centrifuged to collect and obtain an about
8.5 l supernatant as a cell extract. Determination of the cell
extract for trehalose-releasing enzyme activity revealed that the
culture contained about 0.3 unit/ml culture of the enzyme activity.
To the cell extract was added ammonium sulfate to give a saturation
degree of 0.7 to effect salting out, and then centrifuged to obtain
the precipitate. The precipitate was dissolved in 10 mM phosphate
buffer (pH 7.0), and dialyzed against a fresh preparation of the
same buffer. The dialyzed inner solution was subjected to
ion-exchange chromatography using "SEPABEADS FP-DA13 GEL"
commercialized by Mitsubishi Chemical Co., Ltd., Tokyo, Japan, in
accordance with the method in Example 5-2 except that the resin
volume used of the ion exchanger was about two liters, followed by
collecting fractions having an trehalose-releasing enzyme activity.
The fractions were pooled and dialyzed against a fresh preparation
of the same buffer but containing 1 M ammonium sulfate, and then
the dialyzed solution was centrifuged to obtain the formed
supernatant. The supernatant was subjected to a hydrophobic column
chromatography using "BUTYL TOYOPEARL 650M GEL", a hydrophobic gel
commercialized by Tosoh Co., Ltd., Tokyo, Japan, in accordance with
the method in Example 5-2 except that an about 350 ml of the gel
was used, and then fractions with a trehalose-releasing enzyme
activity were collected. The enzyme collected was confirmed to have
an optimum temperature in a medium temperature range, i.e.,
temperatures over 45.degree. C. but below 60.degree. C. and an
optimum pH in an acid pH range, i.e., a pH of less than 7.
Thus an about 6,400 units of the present trehalose-releasing enzyme
was obtained.
EXAMPLE 7-2
Preparation of Enzyme Using Microorganism of the Genus
Arthrobacter
A seed culture of Arthrobacter sp. S34, FERM BP-6450, was
inoculated to a nutrient culture medium in accordance with the
method in Example 7-1. To one l of the resulting culture was added
100 mg "OVALBUMIN LYSOZYME", a lysozyme preparation, commercialized
by Nagase Biochemicals, Ltd., Kyoto, Japan. Then aeration was
suspended, and cells were disrupted by keeping the culture for 24
hours under the same temperature and stirring conditions as used in
the culture. The cell disruptant was subjected to a continuous
centrifuge at 10,000 rpm, followed by collecting a supernatant as a
cell extract. In accordance with the method in Example 7-1, the
cell extract was treated with salting out, and the sediment was
dialyzed. The resulting dialyzed inner solution was subjected to
ion-exchange chromatography using "SEPABEADS FP-DA13 GEL", a
product of Mitsubishi Chemical Co., Ltd., Tokyo, Japan, in
accordance with the method in Example 7-1 to collect fractions with
a trehalose-releasing enzyme activity. The pooled fractions
contained about 16,500 units of the present trehalose-releasing
enzyme and about 5,500 units the present non-reducing
saccharide-forming enzyme. Thus an enzyme preparation containing
the present two types of enzymes was obtained.
EXAMPLE 7-3
Production of Enzyme Using Transformant
In a 500-ml Erlenmeyer flask were placed a 100 ml aqueous solution
containing 16 g/l polypeptone, 10 g/l yeast extract, and 5 g/l
sodium chloride, and the flask was autoclaved at 121.degree. C. for
15 min, cooled, aseptically adjusted to pH 7.0, and aseptically
admixed with 10 mg ampicillin in a sodium salt to obtained a
nutrient culture medium. The transformant "GZ2" obtained in Example
6-2 was inoculated into the liquid medium, followed by the
incubation at 37.degree. C. for about 20 hours under
aeration-agitation conditions to obtain a seed culture. Seven
liters of a fresh preparation of the same medium as used in the
seed culture were similarly prepared and placed in a 10-l
fermenter, inoculated with 70 ml of the seed culture, and cultures
for about 20 hours under aeration-agitation conditions. Cells were
collected by centrifuging the resulting culture in usual mariner.
The collected cells were suspended in 10 mM phosphate buffer (pH
7.0) and ultrasonicated to disrupt the cells. The resulting mixture
was centrifuged to remove insoluble substances, followed by
collecting a supernatant as a cell extract. The cell extract was
dialyzed against 10 mM phosphate buffer (pH 7.0). The dialyzed
inner solution was collected and confirmed to have an optimum
temperature in a medium temperature range, i.e., temperatures over
45.degree. C. but below 60.degree. C. and an optimum pH in an acid
pH range, i.e., a pH of less than 7.
Thus the present trehalose-releasing enzyme was obtained. In this
Example, an about 0.5 unit/ml culture of the trehalose-releasing
enzyme was obtained.
As a control, "XL1-Blue", an Escherichia coli strain commercialized
by Stratagene Cloning Systems, California, USA, was cultured under
the same culture conditions as used in the above in a fresh
preparation of the same culture medium as above but free of
ampicillin, followed by collecting and dialyzing a cell extract
similarly as above. No trehalose-releasing enzyme activity was
observed, meaning that the transformant GZ2 can be advantageously
used in producing the present trehalose-releasing enzyme.
EXAMPLE 7-4
Production of Enzyme Using Transformant
The transformant GZ3 in Example 6-3 was cultured similarly as in
Example 7-3 except for using a liquid nutrient culture medium (pH
7.0) consisting of one w/v % maltose, three w/v % polypeptone, one
w/v % "MEAST PIG" commercialized by Asahi Breweries, Ltd., Tokyo,
Japan, 0.1 w/v % dipotassium hydrogen phosphate, 100 .mu.g/ml
ampicillin, and water. The resulting culture was treated with
ultrasonication to disrupt cells, and the mixture was centrifuged
to remove insoluble substances. Measurement of the
trehalose-releasing enzyme activity in the resulting supernatant
revealed that it contained about 70 units/ml culture of the enzyme.
In accordance with the method in Example 5-2, the supernatant was
purified and confirmed that the purified specimen had an optimum
temperature in a medium temperature range, i.e., temperatures over
45.degree. C. but below 60.degree. C. and an optimum pH in an acid
pH range, i.e., a pH of less than 7. Thus the present
trehalose-releasing enzyme was obtained.
EXAMPLE 7-5
Production of Enzyme Using Transformant
The transformant GZ4 in Example 6-4 was cultured similarly as in
Example 4-4. The resulting culture was treated with ultrasonication
to disrupt cells, and the mixture was centrifuged to remove
insoluble substances. Measurement of the trehalose-releasing enzyme
activity in the resulting supernatant revealed that it contained
about 250 units/ml culture of the enzyme. In accordance with the
method in Example 5-2, the supernatant was purified and confirmed
that the purified specimen had an optimum temperature in a medium
temperature range, i.e., temperatures over 45.degree. C. but below
60.degree. C. and an optimum pH in an acid pH range, i.e., a pH of
less than 7. Thus the present trehalose-releasing enzyme was
obtained.
EXAMPLE 8
Saccharide Production
EXAMPLE 8-1
Production of Non-reducing Saccharide Syrup
A 6 w/w % potato starch suspension was gelatinized by heating,
adjusted to pH 4.5 and 50.degree. C., admixed with 2,500 units/g
starch, d.s.b., and enzymatically reacted for 20 hours. The
reaction mixture was adjusted to pH 6.5, autoclaved at 120.degree.
C. for 10 min, cooled to 40.degree. C., admixed with 150 units/g
starch, d.s.b., of "TERMAMYL 60L", an .alpha.-amylase specimen
commercialized by Novo Nordisk Industri A/S, Copenhagen, Denmark,
and subjected to an enzymatic reaction for 20 hours while keeping
at the temperature. The reaction mixture was autoclaved at
120.degree. C. for 20 min, cooled to 53.degree. C., adjusted to pH
5.7, admixed with one unit per gram starch, d.s.b., of a
non-reducing saccharide-forming enzyme obtained by the method in
Example 4-1, and subjected to an enzymatic reaction for 96 hours.
The reaction mixture thus obtained was heated at 97.degree. C. for
30 min to inactivate the remaining enzyme, cooled, filtered,
purified in a usual manner by decoloration with an activated
charcoal and desalting with ion exchangers, and concentrated to
obtain an about 70 w/w % syrup in a yield of about 90% to the
material starch, d.s.b.
The product, which has a low DE of 24 and contains
.alpha.-glucosyltrehalose, .alpha.-maltosyltrehalose,
.alpha.-maltotriosyltrehalose, .alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose in respective amount of 11.5, 5.7,
29.5, 3.5, and 2.8%, d.s.b., has a mild and high-quality sweetness,
and a satisfactory viscosity and moisture-retaining ability. It can
be arbitrarily used as a sweetener, taste-improving agent,
quality-improving agent, stabilizer, filler, adjuvant or excipient
in compositions in general such as foods, cosmetics, and
pharmaceuticals.
EXAMPLE 8-2
Production of Syrup Containing Non-reducing Saccharide
To a 33 w/w % corn starch suspension was added calcium carbonate to
give a final concentration of 0.1 w/w %, and then the mixture was
adjusted to pH 6.5, admixed with 0.2 w/w % per starch, d.s.b., of
"TERMAMYL 60L", a liquefying .alpha.-amylase specimen
commercialized by Novo Nordisk Industri A/S, Copenhagen, Denmark,
and enzymatically reacted at 95.degree. C. for 15 min to liquefy
the starch. The liquefied starch was autoclaved at 120.degree. C.
for 10 min, cooled to 53.degree. C., admixed with one unit/g
starch, d.s.b., of a maltotetraose-forming enzyme from a
Pseudomonas stutzeri strain commercialized by Hayashibara
Biochemical Laboratories Inc., Okayama, Japan, and two units/g
starch, d.s.b., of a non-reducing saccharide-forming enzyme
obtained by the method in Example 4-2, and enzymatically reacted
for 48 hours. The reaction mixture was admixed with 15 units of
".alpha.-AMYLASE 2A", an .alpha.-amylase specimen commercialized by
Ueda Chemical Co., Ltd., Osaka, Japan, and then incubated at
65.degree. C. for two hours, autoclaved at 120.degree. C. for 10
min, and cooled. The resulting mixture was filtered, and in a usual
manner purified by treatments of coloration using an activated
charcoal and of desalting using ion exchangers, and concentrated
into an about 70 w/w % syrup, d.s.b., in a yield of about 90% with
respect to the material starch, d.s.b.
The product, which has a low DE of 18.5 and contains
.alpha.-glucosyltrehalose, .alpha.-maltosyltrehalose,
.alpha.-maltotriosyltrehalose, .alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose in respective amount of 9.3, 30.1,
0.9, 0.8, and 0.5%, d.s.b., has a mild and high-quality sweetness,
and a satisfactory viscosity and moisture-retaining ability. It can
be arbitrarily used as a sweetener, taste-improving agent,
quality-improving agent, stabilizer, filler, adjuvant or excipient
in compositions in general such as foods, cosmetics, and
pharmaceuticals.
EXAMPLE 8-3
Production of Non-reducing Saccharide
A 20 w/w % aqueous solution of any of reducing partial starch
hydrolyzates of maltotriose, maltotetraose, maltopentaose,
maltohexaose, and maltoheptaose, which are all produced by
Hayashibara Biochemical Laboratories Inc., Okayama, Japan, admixed
with two units/g reducing partial starch hydrolyzate of a purified
specimen of non-reducing saccharide-forming enzyme obtained by the
method in Example 2-2, and subjected to an enzymatic reaction at
50.degree. C. and pH 6.0 for 48 hours. From each of the
above-identified reducing partial starch hydrolyzates were
respectively formed .alpha.-glucosyltrehalose,
.alpha.-maltosyltrehalose, .alpha.-maltotriosyltrehalose,
.alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose as reducing saccharides.
Saccharides in each reaction mixture were in conventional manner
fractionated by the following successive treatments: Inactivation
of the remaining enzyme by heating, filtration, decoloration,
desalting, concentration, and column chromatography using "XT-1016
(Na.sup.+-form)", an alkali-metal strong-acid cation exchange resin
with a polymerization degree of 4%, commercialized by Tokyo Organic
Chemical Industries, Ltd., Tokyo, Japan. The conditions used in the
column chromatography were as follows: The inner column temperature
was set to 55.degree. C., the load volume of a saccharide solution
to the resin was about 5 v/v %, and the flow rate of water heated
to 55.degree. C. as a moving bed was set to SV (space velocity)
0.13. An eluate from each column, which contained at least 95 w/w %
of any of the above-identified non-reducing saccharides, d.s.b.,
with respect to saccharide composition, was collected. To each
collected eluate was added sodium hydroxide to give a concentration
of 0.1 N, and the mixture was heated at 100.degree. C. for two
hours to decompose the remaining reducing saccharides. The reaction
mixtures thus obtained were respectively decolored with an
activated charcoal, desalted with ion exchangers in H-- and
OH-form, concentrated, dried in vacuo, and pulverized into powdery
.alpha.-glucosyltrehalose, .alpha.-maltosyltrehalose,
.alpha.-maltotriosyltrehalose, .alpha.-maltotetraosyltrehalose, and
.alpha.-maltopentaosyltrehalose with a purity of at least 99.0 w/w
%, d.s.b.
The products, containing highly-purified non-reducing saccharides
and having a more lower DE, can be arbitrarily used as a
taste-improving agent, quality-improving agent, stabilizer, filler,
adjuvant or excipient in compositions in general such as foods,
cosmetics, and pharmaceuticals.
EXAMPLE 8-4
Production of Crystalline Powder Containing Non-reducing
Saccharide
An aqueous 20 w/w % solution of maltopentaose commercialized by
Hayashibara Biochemical Laboratories Inc., Okayama, Japan, was
prepared, admixed with two units/g maltopentaose, d.s.b., of a
non-reducing saccharide-forming enzyme obtained by the method in
Example 4-3, and enzymatically reacted at 50.degree. C. for 48
hours, resulting in a conversion of about 75% maltopentaose into
.alpha.-maltotriosyltrehalose. The reaction mixture was heated at
97.degree. C. for 30 min to inactivate the remaining enzyme, and
then cooled, filtered, and purified by decoloration using an
activated charcoal and desalting using ion exchangers.
Thereafter, the resulting solution was concentrated into an about
75 w/w % solution with respect to solid contents, admixed with an
about 0.01 w/v .alpha.-maltotriosyltrehalose crystal as a seed
crystal, and allowed to stand for 24 hours. Then the crystallized
.alpha.-maltotriosyltrehalose crystal was collected by a
centrifuge, washed with a small amount of cold water, and dried in
a usual manner to obtain a crystalline powder with a
relatively-high content of the non-reducing saccharide in a yield
of about 50% to the material solids, d.s.b.
The product, having a relatively-low sweetness and an extremely-low
DE of less than 0.2 and containing at least 99.0 w/w % of
.alpha.-maltotriosyltrehalose as a non-reducing saccharide, can be
arbitrarily used as a taste-improving agent, quality-improving
agent, stabilizer, filler, adjuvant or excipient in compositions in
general such as foods, cosmetics, and pharmaceuticals.
EXAMPLE 8-5
Process for Producing Hydrous Crystalline Trehalose
Corn starch was suspended in water into a 30 w/w % starch
suspension which was then admixed with calcium carbonate in an
amount of 0.1 w/w %. The mixture was adjusted to pH 6.0, and then
admixed with 0.2 w/w % per starch, d.s.b., of "TERMAMYL 60L", a
liquefying .alpha.-amylase specimen commercialized by Novo Nordisk
Industri A/S, Copenhagen, Denmark, and enzymatically reacted at
95.degree. C. for 15 min to gelatinize and liquefy the starch. The
resulting mixture was autoclaved at 120.degree. C. for 30 min,
cooled to 51.degree. C., adjusted to pH 5.7, and enzymatically
reacted at the same temperature for 64 hours after admixed with 300
units/g starch, d.s.b., of an isoamylase specimen commercialized by
Hayashibara Biochemical Laboratories Inc., Okayama, Japan; two
units/g starch, d.s.b., of a cyclomaltodextrin glucanotransferase
specimen commercialized by Hayashibara Biochemical Laboratories
Inc., Okayama, Japan; two units of a non-reducing
saccharide-forming enzyme obtained by the method in Example 4-1;
and 10 unit/g starch, d.s.b., of a trehalose-releasing enzyme
obtained by the method in Example 7-1. The reaction mixture was
heated at 97.degree. C. for 30 min to inactivate the remaining
enzyme, and then cooled 50.degree. C., admixed with 10 unit/g
starch, d.s.b., of "GLUCOZYME", a glucoamylase specimen
commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and
subjected to an enzymatic reaction for 24 hours. The reaction
mixture thus obtained was heated at 95.degree. C. for 10 min to
inactivate the remaining enzymes, cooled, filtered, purified by
decoloration using an activated charcoal and desalting using ion
exchangers, and concentrated to an about 60 w/w % solution with
respect to solid contents or a syrup containing 84.1 w/w %
trehalose, d.s.b. The syrup was concentrated up to give a
concentration of about 83 w/w %, d.s.b., and the concentrate was
placed in a crystallizer, admixed with an about 0.1 w/v % hydrous
crystalline trehalose to the syrup, and stirred for about two hours
to crystallize the saccharide. The resulting crystals were
collected by a centrifuge, washed with a small amount of water to
remove molasses, dried by air heated to 45.degree. C. to obtain
hydrous crystalline trehalose with a purity of at least 99% in a
yield of about 50% to the material starch, d.s.b.
Since the product is substantially free from hygroscopicity and
easily handleable, it can be arbitrarily used as a sweetener,
taste-improving agent, quality-improving agent, stabilizer, filler,
adjuvant or excipient in compositions in general such as foods,
cosmetics, and pharmaceuticals.
EXAMPLE 8-6
Process for Producing Crystalline Powder Containing Anhydrous
Crystalline Trehalose
Using the method in Example 8-5 hydrous crystalline trehalose was
prepared, and the saccharide was dried in vacuo using a jacketed
rotary-vacuum-dryer. The drying was conducted at 90.degree. C. and
300-350 mmHg for about seven hours. After the drying, the above
temperature and pressure were returned to ambient temperature and
normal pressure before collecting the product or a crystalline
powder containing at least 90 w/w % anhydrous crystalline
trehalose, d.s.b.
Since anhydrous crystalline trehalose absorbs moisture in hydrous
matters and changes in itself into hydrous crystalline trehalose,
the product rich in the saccharide can be arbitrarily used as a
non-harmful safe desiccant to dehydrate or dry compositions
including food products, cosmetics and pharmaceuticals, as well as
materials and intermediates thereof. The product with a mild and
high-quality sweetness can be arbitrarily used as a sweetener,
taste-improving agent, quality-improving agent, stabilizer, filler,
adjuvant or excipient in compositions in general such as foods,
cosmetics, and pharmaceuticals.
EXAMPLE 8-7
Process for Producing Trehalose Syrup
A 27 w/w % suspension of tapioca starch was admixed with calcium
carbonate to give a final concentration of 0.1 w/w %, adjusted to
pH 6.0, admixed with 0.2 w/w % per starch, d.s.b., of "TERMAMYL
60L", a liquefying .alpha.-amylase specimen commercialized by Novo
Nordisk Industri A/S, Copenhagen, Denmark, and enzymatically
reacted at 95.degree. C. for 15 min to gelatinize and liquefy the
starch. The resulting mixture was autoclaved at a pressure of 2
kg/cm.sup.2 for 30 min, cooled to 53.degree. C., adjusted to pH
5.7, and enzymatically reacted at the same temperature for 72 hours
after admixed with 500 units/g starch, d.s.b., of "PROMOZYME 200L",
a pullulanase specimen commercialized by Novo Nordisk Industri A/S,
Copenhagen, Denmark; one unit/g starch, d.s.b., of Pseudomonas
stutzeri strain commercialized by Hayashibara Biochemical
Laboratories Inc., Okayama, Japan; about two units/g starch,
d.s.b., of a non-reducing saccharide-forming enzyme and about six
units/g starch, d.s.b., of a trehalose-releasing enzyme, obtained
by the method in Example 7-2. The reaction mixture thus obtained
was heated at 97.degree. C. for 15 min, cooled and filtered to
obtain a filtrate. The filtrate was in a usual manner purified by
decoloration using an activated charcoal and desalting using ion
exchangers, and concentrated to an about 70 w/w % syrup with
respect to solid contents in a yield of about 92% to the material,
d.s.b.
The product, comprising 35.2% trehalose, 3.4%
.alpha.-glucosyltrehalose, 1.8% glucose, 37.2% maltose, 9.1%
maltotriose, and 13.3% oligosaccharides higher than maltotetraose,
has a mild and high-quality sweetness, relatively-lower
reducibility and viscosity, and adequate moisture-retaining
ability; it can be arbitrarily used as a sweetener, taste-improving
agent, quality-improving agent, stabilizer, filler, adjuvant or
excipient in compositions in general such as foods, cosmetics, and
pharmaceuticals.
EXAMPLE 8-8
Process for Producing Crystalline Powder Containing Anhydrous
Crystalline Trehalose
One part by weight of "EX-I", an amylose commercialized by
Hayashibara Biochemical Laboratories Inc., Okayama, Japan, was
dissolved in 15 parts by weight of water by heating, and the
solution was heated to 53.degree. C. and adjusted to pH 5.7. To the
resulting solution was added two units/g amylose, d.s.b., of a
non-reducing saccharide-forming enzyme, obtained in Example 4-3,
and six units/g amylose, d.s.b., of a trehalose-releasing enzyme,
obtained by the method in Example 7-4, followed by an incubation
for 48 hours. The reaction mixture was heated at 97.degree. C. for
30 min to inactivate the remaining enzyme, and then adjusted to pH
5.0, admixed with 10 units/g amylose, d.s.b., of "GLUCOZYME", a
glucoamylase specimen commercialized by Nagase Biochemicals, Ltd.,
Kyoto, and enzymatically reacted for 40 hours. The reaction mixture
thus obtained was heated at 95.degree. C. for 10 min to inactivate
the remaining enzymes, cooled, filtered, purified by decoloration
using an activated charcoal and desalting using ion exchangers, and
concentrated to an about 60 w/w % solution with respect to solid
contents or a syrup containing 82.1 w/w % trehalose, d.s.b.
Similarly as in Example 8-3, the syrup was subjected to column
chromatography, followed by collecting a fraction containing about
98 w/w % trehalose, d.s.b. The fraction was concentrated in vacuo
under heating conditions into an about 85 w/w % syrup with respect
to solid contents. The syrup was admixed with hydrous crystalline
trehalose as a seed crystal in an about 2 w/v % of to the syrup,
stirred at 120.degree. C. for five minutes, distributed to plastic
vats, and dried at 100.degree. C. in vacuo to crystallize the
saccharide. Thereafter, the contents in a block form were detached
from the vats and cut with a cutter to obtain a solid product,
containing anhydrous crystalline trehalose with a crystallinity of
about 70% and having a moisture content of about 0.3 w/w % in a
yield of about 70% to the material amylose, d.s.b. The solid
product was pulverized in a usual manner into a crystalline powdery
containing anhydrous crystalline trehalose.
Since anhydrous crystalline trehalose absorb moisture from hydrous
matters and changes into hydrous crystalline trehalose, the product
rich in anhydrous crystalline trehalose can be arbitrarily used as
a non-harmful safe desiccant to dehydrate or dry compositions
including food products, cosmetics and pharmaceuticals, as well as
materials and intermediates thereof. The product with a mild and
high-quality sweetness can be arbitrarily used as a sweetener,
taste-improving agent, quality-improving agent, stabilizer, filler,
adjuvant or excipient in compositions in general such as foods,
cosmetics, and pharmaceuticals.
As described above, the present invention was made based on the
finding of a novel non-reducing saccharide-forming enzyme and a
novel trehalose-releasing enzyme, which have an optimum temperature
in a medium temperature range and preferably have an optimum pH in
an acid pH range. These enzymes according to the present invention
can be obtained in a desired amount, for example, by culturing
microorganisms capable of producing the enzymes. The present DNAs
which encode either of the enzymes are quite useful in producing
such enzymes as recombinant proteins. In cases of using
transformant introduced with the DNAs, the enzymes according to the
present invention can be yielded in a desired amount. The present
enzymes can be used in producing non-reducing saccharides having a
trehalose structure, which include trehalose, in a medium
temperature rang and/or an acid pH range. Particularly, when used
the present enzymes in combination with other saccharide-related
enzymes having an optimum temperature in a medium temperature rang
and/or an optimum pH in an acid pH range, desired saccharides can
be produced quite efficiently. The enzymes according to the present
invention are ones with revealed amino acid sequences; they can be
safely used to produce the non-reducing saccharides to be used in
food products and pharmaceuticals. The non-reducing saccharides and
reducing saccharides, which contain the same and have a lesser
reducibility, produced by the present invention have a mild and
high-quality sweetness, and most preferably have an insubstantial
reducibility or a reduced reducibility by a large margin.
Therefore, the saccharides can be arbitrarily used as in
compositions in general such as foods, cosmetics, and
pharmaceuticals with lesser fear of coloration and
deterioration.
The present invention with these unfathomable advantageous
properties and features is a useful invention that would greatly
contribute to this art.
While there has been described what is at present considered to be
the preferred embodiments of the invention, it will be understood
that various modifications may be made therein, and it is intended
to cover in the appended claims all such modifications as fall
within the true spirit and scope of the invention.
SEQUENCE LISTINGS
1
39 1 756 PRT ARTHROBACTER sp.S34 1 Pro Ala Ser Thr Tyr Arg Leu Gln
Ile Ser Ala Glu Phe Thr Leu Phe 1 5 10 15 Asp Ala Ala Arg Ile Val
Pro Tyr Leu His Arg Leu Gly Ala Asp Trp 20 25 30 Leu Tyr Leu Ser
Pro Leu Leu Glu Ser Glu Ser Gly Ser Ser His Gly 35 40 45 Tyr Asp
Val Val Asp His Ser Arg Val Asp Ala Ala Arg Gly Gly Pro 50 55 60
Glu Gly Leu Ala Glu Leu Ser Arg Ala Ala His Glu Arg Gly Met Gly 65
70 75 80 Val Val Val Asp Ile Val Pro Asn His Val Gly Val Ala Thr
Pro Lys 85 90 95 Ala Asn Arg Trp Trp Trp Asp Val Leu Ala Arg Gly
Gln Arg Ser Glu 100 105 110 Tyr Ala Asp Tyr Phe Asp Ile Asp Trp Glu
Phe Gly Gly Gly Arg Leu 115 120 125 Arg Leu Pro Val Leu Gly Asp Gly
Pro Asp Glu Leu Asp Ala Leu Arg 130 135 140 Val Asp Gly Asp Glu Leu
Val Tyr Tyr Glu His Arg Phe Pro Ile Ala 145 150 155 160 Glu Gly Thr
Gly Gly Gly Thr Pro Arg Glu Val His Asp Arg Gln His 165 170 175 Tyr
Glu Leu Met Ser Trp Arg Arg Ala Asp His Asp Leu Asn Tyr Arg 180 185
190 Arg Phe Phe Ala Val Asn Thr Leu Ala Ala Val Arg Val Glu Asp Pro
195 200 205 Arg Val Phe Asp Asp Thr His Arg Glu Ile Gly Arg Trp Ile
Ala Glu 210 215 220 Gly Leu Val Asp Gly Leu Arg Val Asp His Pro Asp
Gly Leu Arg Ala 225 230 235 240 Pro Gly Asp Tyr Leu Arg Arg Leu Ala
Glu Leu Ala Gln Gly Arg Pro 245 250 255 Ile Trp Val Glu Lys Ile Ile
Glu Gly Asp Glu Arg Met Pro Pro Gln 260 265 270 Trp Pro Ile Ala Gly
Thr Thr Gly Tyr Asp Ala Leu Ala Gly Ile Asp 275 280 285 Arg Val Leu
Val Asp Pro Ala Gly Glu His Pro Leu Thr Gln Ile Val 290 295 300 Asp
Glu Ala Ala Gly Ser Pro Arg Arg Trp Ala Glu Leu Val Pro Glu 305 310
315 320 Arg Lys Arg Ala Val Ala Arg Gly Ile Leu Asn Ser Glu Ile Arg
Arg 325 330 335 Val Ala Arg Glu Leu Gly Glu Val Ala Gly Asp Val Glu
Asp Ala Leu 340 345 350 Val Glu Ile Ala Ala Ala Leu Ser Val Tyr Arg
Ser Tyr Leu Pro Phe 355 360 365 Gly Arg Glu His Leu Asp Glu Ala Val
Ala Ala Ala Gln Ala Ala Ala 370 375 380 Pro Gln Leu Glu Ala Asp Leu
Ala Ala Val Gly Ala Ala Leu Ala Asp 385 390 395 400 Pro Gly Asn Pro
Ala Ala Leu Arg Phe Gln Gln Thr Ser Gly Met Ile 405 410 415 Met Ala
Lys Gly Val Glu Asp Asn Ala Phe Tyr Arg Tyr Pro Arg Leu 420 425 430
Thr Ser Leu Thr Glu Val Gly Gly Asp Pro Ser Leu Phe Ala Ile Asp 435
440 445 Ala Ala Ala Phe His Ala Ala Gln Arg Asp Arg Ala Ala Arg Leu
Pro 450 455 460 Glu Ser Met Thr Thr Leu Thr Thr His Asp Thr Lys Arg
Ser Glu Asp 465 470 475 480 Thr Arg Ala Arg Ile Thr Ala Leu Ala Glu
Ala Pro Glu Arg Trp Arg 485 490 495 Arg Phe Leu Thr Glu Val Gly Gly
Leu Ile Gly Thr Gly Asp Arg Val 500 505 510 Leu Glu Asn Leu Ile Trp
Gln Ala Ile Val Gly Ala Trp Pro Ala Ser 515 520 525 Arg Glu Arg Leu
Glu Ala Tyr Ala Leu Lys Ala Ala Arg Glu Ala Gly 530 535 540 Glu Ser
Thr Asp Trp Ile Asp Gly Asp Pro Ala Phe Glu Glu Arg Leu 545 550 555
560 Thr Arg Leu Val Thr Val Ala Val Glu Glu Pro Leu Val His Glu Leu
565 570 575 Leu Glu Arg Leu Val Asp Glu Leu Thr Ala Ala Gly Tyr Ser
Asn Gly 580 585 590 Leu Ala Ala Lys Leu Leu Gln Leu Leu Ala Pro Gly
Thr Pro Asp Val 595 600 605 Tyr Gln Gly Thr Glu Arg Trp Asp Arg Ser
Leu Val Asp Pro Asp Asn 610 615 620 Arg Arg Pro Val Asp Phe Ala Ala
Ala Ser Glu Leu Leu Asp Arg Leu 625 630 635 640 Asp Gly Gly Trp Arg
Pro Pro Val Asp Glu Thr Gly Ala Val Lys Thr 645 650 655 Leu Val Val
Ser Arg Ala Leu Arg Leu Arg Arg Asp Arg Pro Glu Leu 660 665 670 Phe
Thr Ala Tyr His Pro Val Thr Ala Arg Gly Ala Gln Ala Glu His 675 680
685 Leu Ile Gly Phe Asp Arg Gly Gly Ala Ile Ala Leu Ala Thr Arg Leu
690 695 700 Pro Leu Gly Leu Ala Ala Ala Gly Gly Trp Gly Asp Thr Val
Val Asp 705 710 715 720 Val Gly Glu Arg Ser Leu Arg Asp Glu Leu Thr
Gly Arg Glu Ala Arg 725 730 735 Gly Ala Ala Arg Val Ala Glu Leu Phe
Ala Asp Tyr Pro Val Ala Leu 740 745 750 Leu Val Glu Thr 755 2 6 PRT
ARTHROBACTER sp.S34 2 Asp Ile Val Pro Asn His 1 5 3 6 PRT
ARTHROBACTER sp.S34 3 Gly Thr Thr Gly Tyr Asp 1 5 4 20 PRT
ARTHROBACTER sp.S34 4 Pro Ala Ser Thr Tyr Arg Leu Gln Ile Ser Ala
Glu Phe Thr Leu Phe 1 5 10 15 Asp Ala Ala Arg 20 5 20 PRT
ARTHROBACTER sp.S34 5 Ser Leu Val Asp Pro Asp Asn Arg Arg Pro Val
Asp Phe Ala Ala Ala 1 5 10 15 Ser Glu Leu Leu 20 6 20 PRT
ARTHROBACTER sp.S34 6 Ala Asn Arg Trp Trp Trp Asp Val Leu Ala Arg
Gly Gln Arg Ser Glu 1 5 10 15 Tyr Ala Asp Tyr 20 7 2268 DNA
ARTHROBACTER sp.S34 7 cccgccagta cctaccgcct tcagatctcg gcggagttca
ccctcttcga cgcggcgcgc 60 atcgtgccct acctgcaccg cctcggcgcc
gactggctgt acctctcgcc gctgctcgag 120 tccgagtcgg gctcctcgca
cggctacgac gtggtcgacc actcccgcgt cgacgccgcc 180 cgcggcgggc
cggaggggct cgccgagctc tcccgtgcgg cgcacgagcg cggcatgggc 240
gtcgtcgtcg acatcgtgcc caaccacgtc ggcgtcgcga cgccgaaggc gaaccgctgg
300 tggtgggacg ttctggcccg tggacagcgg tcggagtacg ccgactactt
cgacatcgac 360 tgggagttcg gcggcggcag gctgcgcctg cccgtgctcg
gcgacggccc cgacgagctc 420 gacgcgctga gagtggatgg cgacgagctc
gtctactacg agcaccgctt cccgatcgcc 480 gagggcaccg gcggcggcac
cccgcgcgag gtgcacgacc ggcagcacta cgagctgatg 540 tcgtggcggc
gggccgacca cgacctcaac taccgccgct tcttcgccgt gaacacgctc 600
gccgccgtac gcgtcgaaga cccgcgcgtg ttcgacgaca cccaccgcga gatcggccgc
660 tggatcgccg agggcctcgt cgacggcctg cgcgtcgacc accccgacgg
gctgcgcgcc 720 cccggcgact acctgcgccg tctcgccgag ctcgcccaag
gcaggccgat ctgggtcgag 780 aagatcatcg agggcgacga gcggatgccc
ccgcagtggc ccatcgccgg caccaccggc 840 tacgacgcgc tggccgggat
cgaccgggtg ctcgtcgacc ccgcgggcga gcatccgctc 900 acccagatcg
tcgacgaggc ggcaggcagc ccccggcgct gggccgagct ggttcccgag 960
cgcaagcggg ccgtcgcccg cggcatcctg aactccgaga tccgccgcgt cgcccgcgaa
1020 ctcggagagg tcgccggcga cgtcgaagac gcgctcgtcg agatcgccgc
cgccctgtcc 1080 gtctaccgca gctacctgcc gttcgggcgc gagcacctcg
acgaagccgt ggccgccgcg 1140 caggccgcag ccccccagct cgaggccgac
ctcgccgccg tcggcgcagc gctcgccgac 1200 ccgggcaacc ccgccgcgct
ccgcttccag cagaccagcg gcatgatcat ggccaagggc 1260 gtcgaggaca
acgcgttcta ccgctacccc cggctcacct cgctgaccga ggtcggggga 1320
gacccgagcc tgttcgcgat cgacgcggcc gccttccacg cggcgcagcg cgaccgcgcc
1380 gcccggctgc ccgagtcgat gacgacgctg accacccacg acaccaagcg
cagcgaagac 1440 acccgggcgc ggatcaccgc gctcgccgag gcccccgaac
gctggcggcg cttcctgacc 1500 gaggtcggcg ggctcatcgg aacgggcgac
cgggtgctgg agaacctgat ctggcaggcg 1560 atcgtcggcg cgtggccggc
gagccgggag cggctcgagg cctacgcgct gaaggccgcg 1620 cgcgaagccg
gcgagtcgac cgactggatc gacggcgacc ccgcgttcga agagcggctg 1680
acccgcctgg tcacggtcgc cgtcgaggag ccgctcgtgc acgagctgct cgagcggctc
1740 gtcgacgagc tgacggcggc cgggtactcc aacggcctcg cggcgaagct
gctgcagctg 1800 ctcgcccccg gaacccccga cgtgtaccag ggcacggaac
gctgggaccg gtcgctggtg 1860 gacccggaca accgtcgccc cgtggatttc
gccgcggcat ccgagctgct cgaccgcctc 1920 gacggcggct ggcggccgcc
cgtcgacgag accggcgcgg tcaagacgct cgtcgtctcc 1980 cgcgcgctgc
ggctgcgccg cgaccggccc gagctgttca ccgcgtacca cccggtcacg 2040
gcgcgcggcg cgcaggccga gcacctgatc ggcttcgacc gcggcggcgc gatcgccctg
2100 gccacccgcc tgccgctcgg cctcgccgcc gcaggcggct ggggcgacac
ggtcgtcgac 2160 gtcggcgagc ggagcctgcg cgacgagctg accggccgcg
aggcccgcgg agcggcgcgc 2220 gtggccgagt tgttcgccga ctaccccgtc
gccctgctgg tggagaca 2268 8 28 DNA ARTHROBACTER sp.S34 8 ttttttaata
aaatcaggag gaaaaaat 28 9 575 PRT ARTHROBACTER sp.S34 9 Met Asn Arg
Arg Phe Pro Val Trp Ala Pro Gln Ala Ala Gln Val Thr 1 5 10 15 Leu
Val Val Gly Gln Gly Arg Ala Glu Leu Pro Leu Thr Arg Asp Glu 20 25
30 Asn Gly Trp Trp Ala Leu Gln Gln Pro Trp Asp Gly Gly Pro Asp Leu
35 40 45 Val Asp Tyr Gly Tyr Leu Val Asp Gly Lys Gly Pro Phe Ala
Asp Pro 50 55 60 Arg Ser Leu Arg Gln Pro Arg Gly Val His Glu Leu
Gly Arg Glu Phe 65 70 75 80 Asp Pro Ala Arg Tyr Ala Trp Gly Asp Asp
Gly Trp Arg Gly Arg Asp 85 90 95 Leu Thr Gly Ala Val Ile Tyr Glu
Leu His Val Gly Thr Phe Thr Pro 100 105 110 Glu Gly Thr Leu Asp Ser
Ala Ile Arg Arg Leu Asp His Leu Val Arg 115 120 125 Leu Gly Val Asp
Ala Val Glu Leu Leu Pro Val Asn Ala Phe Asn Gly 130 135 140 Thr His
Gly Trp Gly Tyr Asp Gly Val Leu Trp Tyr Ala Val His Glu 145 150 155
160 Pro Tyr Gly Gly Pro Glu Ala Tyr Gln Arg Phe Val Asp Ala Cys His
165 170 175 Ala Arg Gly Leu Ala Val Val Gln Asp Val Val Tyr Asn His
Leu Gly 180 185 190 Pro Ser Gly Asn His Leu Pro Asp Phe Gly Pro Tyr
Leu Gly Ser Gly 195 200 205 Ala Ala Asn Thr Trp Gly Asp Ala Leu Asn
Leu Asp Gly Pro Leu Ser 210 215 220 Asp Glu Val Arg Arg Tyr Ile Ile
Asp Asn Ala Val Tyr Trp Leu Arg 225 230 235 240 Asp Met His Ala Asp
Gly Leu Arg Leu Asp Ala Val His Ala Leu Arg 245 250 255 Asp Ala Arg
Ala Leu His Leu Leu Glu Glu Leu Ala Ala Arg Val Asp 260 265 270 Glu
Leu Ala Gly Glu Leu Gly Arg Pro Leu Thr Leu Ile Ala Glu Ser 275 280
285 Asp Leu Asn Asp Pro Lys Leu Ile Arg Ser Arg Ala Ala His Gly Tyr
290 295 300 Gly Leu Asp Ala Gln Trp Asp Asp Asp Val His His Ala Val
His Ala 305 310 315 320 Asn Val Thr Gly Glu Thr Val Gly Tyr Tyr Ala
Asp Phe Gly Gly Leu 325 330 335 Gly Ala Leu Val Lys Val Phe Gln Arg
Gly Trp Phe His Asp Gly Thr 340 345 350 Trp Ser Ser Phe Arg Glu Arg
His His Gly Arg Pro Leu Asp Pro Asp 355 360 365 Ile Pro Phe Arg Arg
Leu Val Ala Phe Ala Gln Asp His Asp Gln Val 370 375 380 Gly Asn Arg
Ala Val Gly Asp Arg Met Ser Ala Gln Val Gly Glu Gly 385 390 395 400
Ser Leu Ala Ala Ala Ala Ala Leu Val Leu Leu Gly Pro Phe Thr Pro 405
410 415 Met Leu Phe Met Gly Glu Glu Trp Gly Ala Arg Thr Pro Trp Gln
Phe 420 425 430 Phe Thr Ser His Pro Glu Pro Glu Leu Gly Glu Ala Thr
Ala Arg Gly 435 440 445 Arg Ile Ala Glu Phe Ala Arg Met Gly Trp Asp
Pro Ala Val Val Pro 450 455 460 Asp Pro Gln Asp Pro Ala Thr Phe Ala
Arg Ser His Leu Asp Trp Ser 465 470 475 480 Glu Pro Glu Arg Glu Pro
His Ala Gly Leu Leu Ala Phe Tyr Thr Asp 485 490 495 Leu Ile Ala Leu
Arg Arg Glu Leu Pro Val Asp Ala Pro Ala Arg Glu 500 505 510 Val Asp
Ala Asp Glu Ala Arg Gly Val Phe Ala Phe Ser Arg Gly Pro 515 520 525
Leu Arg Val Thr Val Ala Leu Arg Pro Gly Pro Val Gly Val Pro Glu 530
535 540 His Gly Gly Leu Val Leu Ala Tyr Gly Glu Val Arg Ala Gly Ala
Ala 545 550 555 560 Gly Leu His Leu Asp Gly Pro Gly Ala Ala Ile Val
Arg Leu Glu 565 570 575 10 6 PRT ARTHROBACTER sp.S34 10 Trp Gly Tyr
Asp Gly Val 1 5 11 6 PRT ARTHROBACTER sp.S34 11 Asp Val Val Tyr Asn
His 1 5 12 7 PRT ARTHROBACTER sp.S34 12 Arg Leu Asp Ala Val His Ala
1 5 13 7 PRT ARTHROBACTER sp.S34 13 Ile Ala Glu Ser Asp Leu Asn 1 5
14 20 PRT ARTHROBACTER sp.S34 14 Met Asn Arg Arg Phe Pro Val Trp
Ala Pro Gln Ala Ala Gln Val Thr 1 5 10 15 Leu Val Val Gly 20 15 20
PRT ARTHROBACTER sp.S34 15 Ser Arg Ala Ala His Gly Tyr Gly Leu Asp
Ala Gln Trp Asp Asp Asp 1 5 10 15 Val His His Ala 20 16 20 PRT
ARTHROBACTER sp.S34 16 Asp Glu Asn Gly Trp Trp Ala Leu Gln Gln Pro
Trp Asp Gly Gly Pro 1 5 10 15 Asp Leu Val Asp 20 17 1725 DNA
ARTHROBACTER sp.S34 17 atgaaccgac gattcccggt ctgggcgccc caggccgcgc
aggtgacgct cgtcgtgggc 60 caaggccgcg ccgaactccc gctgacccgc
gacgagaacg gatggtgggc tcttcagcag 120 ccgtgggacg gcggccccga
cctcgtcgac tacggctacc tcgtcgacgg caagggcccc 180 ttcgccgacc
cgcggtcgct gcggcagccg cgcggcgtgc acgagctcgg ccgcgaattc 240
gaccccgccc gctacgcgtg gggcgacgac ggatggcgcg gccgagacct caccggagcc
300 gtgatctacg aactgcacgt cggcaccttc acccctgagg gaacgctgga
cagcgccatc 360 cgtcgcctcg accacctggt gcgcctcggc gtcgacgcgg
tcgagctgct gcccgtcaac 420 gcgttcaacg gcacccacgg ctggggctac
gacggggtgc tctggtacgc ggtgcacgag 480 ccctacggcg gcccggaggc
gtaccagcgc ttcgtcgacg cctgccacgc ccgcggcctc 540 gccgtcgtgc
aggacgtcgt ctacaaccac ctgggcccga gcggcaacca cctgcccgac 600
ttcggcccct acctcgggtc gggcgccgcc aacacctggg gcgacgcgct gaacctcgac
660 gggccgctct ccgacgaggt gcggcggtac atcatcgaca acgcggtgta
ctggctgcgc 720 gacatgcacg ccgacgggct gcggctcgac gccgtgcacg
cgctgcgcga cgcccgcgcg 780 ctgcacctgc tcgaagagct cgccgcccgc
gtcgacgagc tggcgggcga gctcggccgg 840 ccgctgacgc tcatcgccga
gagcgacctg aacgacccga agctgatccg ctcccgcgcg 900 gcgcacggct
acggcctcga cgcccagtgg gacgacgacg tgcaccacgc ggtgcacgcc 960
aacgtgaccg gcgagaccgt cggctactac gccgacttcg gcgggctcgg cgccctcgtc
1020 aaggtgttcc agcgcggctg gttccacgac ggcacctggt cgagcttccg
cgagcggcac 1080 cacggccggc cgctcgaccc cgacatcccg ttccgccggc
tcgtcgcctt cgcgcaggat 1140 cacgaccagg tcggcaaccg agcggtcggc
gaccgcatgt cggcgcaggt cggcgagggt 1200 tcgctcgccg ccgcggcggc
gctcgtgctg ctcggcccgt tcaccccgat gctgttcatg 1260 ggcgaggagt
ggggcgcgcg caccccgtgg cagttcttca cctcccaccc cgagcccgag 1320
ctgggggagg cgacggcgcg cgggcgcatc gccgagttcg cccgcatggg ctgggacccg
1380 gcagtcgtgc ccgacccgca ggacccggcc accttcgccc gctcgcacct
ggactggtcc 1440 gagcccgagc gggaaccgca cgcgggcctg ctcgccttct
acaccgacct gatcgcgctg 1500 cggcgcgagc tgccggtcga tgcgccggcg
cgcgaggtgg atgccgacga ggcgcgcggc 1560 gtcttcgcgt tcagccgcgg
cccgctgcgg gtcacggtcg cgctgcgccc cggaccggtc 1620 ggggtgcccg
agcacggggg cctcgtgctc gcctacggcg aggtgcgcgc cggcgccgcc 1680
ggactgcacc tcgacgggcc gggagccgcg atcgtgcgcc tcgag 1725 18 23 DNA
ARTHROBACTER sp.S34 18 gcsaaccgst ggtggtggga cgt 23 19 3252 DNA
ARTHROBACTER sp.S34 5'UTR (1)..(742) 3'UTR (742)..(3014) CDS
(743)..(3013) 19 atgccgacga cgaacttgag cgcgttctcg ggcacccgcg
agagcggtcc gcgcacggcg 60 gcgcccagtg ccacgacgag cacgatcgcg
gcgagcgccg cgacgacggc gaccggcagg 120 cgcccctgat tgctggcgaa
ggtgagcacg atgaagacca cctcgaggcc ctcgagcaac 180 acacctttga
acgacacggt gaacgcgtac caatcggaga ccccgaaccg gctctcgcgc 240
cgggcgctct cggccgcctc gacctgacgc cggaaggcag cctcctcgtc acggagagcc
300 ctgcgccctg ccgcgcgcag caccgccttg cgcagccagc cgagcccgaa
gacgagcagc 360 aacccgccga cgacgaggcg cagcacggcc agcggcagca
gcaggatcgc gggaccgacg 420 agcgcgacgg ccgcggccag caccaccacg
gcgacggcgg cacctgtcag cgccgaccgc 480 cagctgcggg tggcgccgac
cgcgacgacg atcgtggtcg cctccaccgc ctcgaccacg 540 caggcgagga
acacggcggc gaacagggcg acggcggtca tcggcccagc agacggttga 600
ccatcacggc acgctagcgc cattgctcac aggaagggcc aagacgcccg caacgcggca
660 cccgtggacg gcgcgtaccg gcgtgtgacc gatcgtgtca accggtggcg
cccgccccga 720 gcacctgcgt agattcggcc tc gtg ccc gcc agt acc tac cgc
ctt cag atc 772 Val Pro Ala Ser Thr Tyr Arg Leu Gln Ile 1 5 10 tcg
gcg gag ttc acc ctc ttc gac gcg gcg cgc atc gtg ccc tac ctg 820 Ser
Ala Glu Phe Thr Leu Phe Asp Ala Ala Arg Ile Val Pro Tyr Leu 15 20
25 cac cgc ctc ggc gcc gac tgg ctg tac ctc tcg ccg ctg ctc gag tcc
868 His Arg Leu Gly Ala Asp Trp Leu Tyr Leu Ser Pro Leu Leu Glu Ser
30
35 40 gag tcg ggc tcc tcg cac ggc tac gac gtg gtc gac cac tcc cgc
gtc 916 Glu Ser Gly Ser Ser His Gly Tyr Asp Val Val Asp His Ser Arg
Val 45 50 55 gac gcc gcc cgc ggc ggg ccg gag ggg ctc gcc gag ctc
tcc cgt gcg 964 Asp Ala Ala Arg Gly Gly Pro Glu Gly Leu Ala Glu Leu
Ser Arg Ala 60 65 70 gcg cac gag cgc ggc atg ggc gtc gtc gtc gac
atc gtg ccc aac cac 1012 Ala His Glu Arg Gly Met Gly Val Val Val
Asp Ile Val Pro Asn His 75 80 85 90 gtc ggc gtc gcg acg ccg aag gcg
aac cgc tgg tgg tgg gac gtt ctg 1060 Val Gly Val Ala Thr Pro Lys
Ala Asn Arg Trp Trp Trp Asp Val Leu 95 100 105 gcc cgt gga cag cgg
tcg gag tac gcc gac tac ttc gac atc gac tgg 1108 Ala Arg Gly Gln
Arg Ser Glu Tyr Ala Asp Tyr Phe Asp Ile Asp Trp 110 115 120 gag ttc
ggc ggc ggc agg ctg cgc ctg ccc gtg ctc ggc gac ggc ccc 1156 Glu
Phe Gly Gly Gly Arg Leu Arg Leu Pro Val Leu Gly Asp Gly Pro 125 130
135 gac gag ctc gac gcg ctg aga gtg gat ggc gac gag ctc gtc tac tac
1204 Asp Glu Leu Asp Ala Leu Arg Val Asp Gly Asp Glu Leu Val Tyr
Tyr 140 145 150 gag cac cgc ttc ccg atc gcc gag ggc acc ggc ggc ggc
acc ccg cgc 1252 Glu His Arg Phe Pro Ile Ala Glu Gly Thr Gly Gly
Gly Thr Pro Arg 155 160 165 170 gag gtg cac gac cgg cag cac tac gag
ctg atg tcg tgg cgg cgg gcc 1300 Glu Val His Asp Arg Gln His Tyr
Glu Leu Met Ser Trp Arg Arg Ala 175 180 185 gac cac gac ctc aac tac
cgc cgc ttc ttc gcc gtg aac acg ctc gcc 1348 Asp His Asp Leu Asn
Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala 190 195 200 gcc gta cgc
gtc gaa gac ccg cgc gtg ttc gac gac acc cac cgc gag 1396 Ala Val
Arg Val Glu Asp Pro Arg Val Phe Asp Asp Thr His Arg Glu 205 210 215
atc ggc cgc tgg atc gcc gag ggc ctc gtc gac ggc ctg cgc gtc gac
1444 Ile Gly Arg Trp Ile Ala Glu Gly Leu Val Asp Gly Leu Arg Val
Asp 220 225 230 cac ccc gac ggg ctg cgc gcc ccc ggc gac tac ctg cgc
cgt ctc gcc 1492 His Pro Asp Gly Leu Arg Ala Pro Gly Asp Tyr Leu
Arg Arg Leu Ala 235 240 245 250 gag ctc gcc caa ggc agg ccg atc tgg
gtc gag aag atc atc gag ggc 1540 Glu Leu Ala Gln Gly Arg Pro Ile
Trp Val Glu Lys Ile Ile Glu Gly 255 260 265 gac gag cgg atg ccc ccg
cag tgg ccc atc gcc ggc acc acc ggc tac 1588 Asp Glu Arg Met Pro
Pro Gln Trp Pro Ile Ala Gly Thr Thr Gly Tyr 270 275 280 gac gcg ctg
gcc ggg atc gac cgg gtg ctc gtc gac ccc gcg ggc gag 1636 Asp Ala
Leu Ala Gly Ile Asp Arg Val Leu Val Asp Pro Ala Gly Glu 285 290 295
cat ccg ctc acc cag atc gtc gac gag gcg gca ggc agc ccc cgg cgc
1684 His Pro Leu Thr Gln Ile Val Asp Glu Ala Ala Gly Ser Pro Arg
Arg 300 305 310 tgg gcc gag ctg gtt ccc gag cgc aag cgg gcc gtc gcc
cgc ggc atc 1732 Trp Ala Glu Leu Val Pro Glu Arg Lys Arg Ala Val
Ala Arg Gly Ile 315 320 325 330 ctg aac tcc gag atc cgc cgc gtc gcc
cgc gaa ctc gga gag gtc gcc 1780 Leu Asn Ser Glu Ile Arg Arg Val
Ala Arg Glu Leu Gly Glu Val Ala 335 340 345 ggc gac gtc gaa gac gcg
ctc gtc gag atc gcc gcc gcc ctg tcc gtc 1828 Gly Asp Val Glu Asp
Ala Leu Val Glu Ile Ala Ala Ala Leu Ser Val 350 355 360 tac cgc agc
tac ctg ccg ttc ggg cgc gag cac ctc gac gaa gcc gtg 1876 Tyr Arg
Ser Tyr Leu Pro Phe Gly Arg Glu His Leu Asp Glu Ala Val 365 370 375
gcc gcc gcg cag gcc gca gcc ccc cag ctc gag gcc gac ctc gcc gcc
1924 Ala Ala Ala Gln Ala Ala Ala Pro Gln Leu Glu Ala Asp Leu Ala
Ala 380 385 390 gtc ggc gca gcg ctc gcc gac ccg ggc aac ccc gcc gcg
ctc cgc ttc 1972 Val Gly Ala Ala Leu Ala Asp Pro Gly Asn Pro Ala
Ala Leu Arg Phe 395 400 405 410 cag cag acc agc ggc atg atc atg gcc
aag ggc gtc gag gac aac gcg 2020 Gln Gln Thr Ser Gly Met Ile Met
Ala Lys Gly Val Glu Asp Asn Ala 415 420 425 ttc tac cgc tac ccc cgg
ctc acc tcg ctg acc gag gtc ggg gga gac 2068 Phe Tyr Arg Tyr Pro
Arg Leu Thr Ser Leu Thr Glu Val Gly Gly Asp 430 435 440 ccg agc ctg
ttc gcg atc gac gcg gcc gcc ttc cac gcg gcg cag cgc 2116 Pro Ser
Leu Phe Ala Ile Asp Ala Ala Ala Phe His Ala Ala Gln Arg 445 450 455
gac cgc gcc gcc cgg ctg ccc gag tcg atg acg acg ctg acc acc cac
2164 Asp Arg Ala Ala Arg Leu Pro Glu Ser Met Thr Thr Leu Thr Thr
His 460 465 470 gac acc aag cgc agc gaa gac acc cgg gcg cgg atc acc
gcg ctc gcc 2212 Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile
Thr Ala Leu Ala 475 480 485 490 gag gcc ccc gaa cgc tgg cgg cgc ttc
ctg acc gag gtc ggc ggg ctc 2260 Glu Ala Pro Glu Arg Trp Arg Arg
Phe Leu Thr Glu Val Gly Gly Leu 495 500 505 atc gga acg ggc gac cgg
gtg ctg gag aac ctg atc tgg cag gcg atc 2308 Ile Gly Thr Gly Asp
Arg Val Leu Glu Asn Leu Ile Trp Gln Ala Ile 510 515 520 gtc ggc gcg
tgg ccg gcg agc cgg gag cgg ctc gag gcc tac gcg ctg 2356 Val Gly
Ala Trp Pro Ala Ser Arg Glu Arg Leu Glu Ala Tyr Ala Leu 525 530 535
aag gcc gcg cgc gaa gcc ggc gag tcg acc gac tgg atc gac ggc gac
2404 Lys Ala Ala Arg Glu Ala Gly Glu Ser Thr Asp Trp Ile Asp Gly
Asp 540 545 550 ccc gcg ttc gaa gag cgg ctg acc cgc ctg gtc acg gtc
gcc gtc gag 2452 Pro Ala Phe Glu Glu Arg Leu Thr Arg Leu Val Thr
Val Ala Val Glu 555 560 565 570 gag ccg ctc gtg cac gag ctg ctc gag
cgg ctc gtc gac gag ctg acg 2500 Glu Pro Leu Val His Glu Leu Leu
Glu Arg Leu Val Asp Glu Leu Thr 575 580 585 gcg gcc ggg tac tcc aac
ggc ctc gcg gcg aag ctg ctg cag ctg ctc 2548 Ala Ala Gly Tyr Ser
Asn Gly Leu Ala Ala Lys Leu Leu Gln Leu Leu 590 595 600 gcc ccc gga
acc ccc gac gtg tac cag ggc acg gaa cgc tgg gac cgg 2596 Ala Pro
Gly Thr Pro Asp Val Tyr Gln Gly Thr Glu Arg Trp Asp Arg 605 610 615
tcg ctg gtg gac ccg gac aac cgt cgc ccc gtg gat ttc gcc gcg gca
2644 Ser Leu Val Asp Pro Asp Asn Arg Arg Pro Val Asp Phe Ala Ala
Ala 620 625 630 tcc gag ctg ctc gac cgc ctc gac ggc ggc tgg cgg ccg
ccc gtc gac 2692 Ser Glu Leu Leu Asp Arg Leu Asp Gly Gly Trp Arg
Pro Pro Val Asp 635 640 645 650 gag acc ggc gcg gtc aag acg ctc gtc
gtc tcc cgc gcg ctg cgg ctg 2740 Glu Thr Gly Ala Val Lys Thr Leu
Val Val Ser Arg Ala Leu Arg Leu 655 660 665 cgc cgc gac cgg ccc gag
ctg ttc acc gcg tac cac ccg gtc acg gcg 2788 Arg Arg Asp Arg Pro
Glu Leu Phe Thr Ala Tyr His Pro Val Thr Ala 670 675 680 cgc ggc gcg
cag gcc gag cac ctg atc ggc ttc gac cgc ggc ggc gcg 2836 Arg Gly
Ala Gln Ala Glu His Leu Ile Gly Phe Asp Arg Gly Gly Ala 685 690 695
atc gcc ctg gcc acc cgc ctg ccg ctc ggc ctc gcc gcc gca ggc ggc
2884 Ile Ala Leu Ala Thr Arg Leu Pro Leu Gly Leu Ala Ala Ala Gly
Gly 700 705 710 tgg ggc gac acg gtc gtc gac gtc ggc gag cgg agc ctg
cgc gac gag 2932 Trp Gly Asp Thr Val Val Asp Val Gly Glu Arg Ser
Leu Arg Asp Glu 715 720 725 730 ctg acc ggc cgc gag gcc cgc gga gcg
gcg cgc gtg gcc gag ttg ttc 2980 Leu Thr Gly Arg Glu Ala Arg Gly
Ala Ala Arg Val Ala Glu Leu Phe 735 740 745 gcc gac tac ccc gtc gcc
ctg ctg gtg gag aca tgaaccgacg attcccggtc 3033 Ala Asp Tyr Pro Val
Ala Leu Leu Val Glu Thr 750 755 tgggcgcccc aggccgcgca ggtgacgctc
gtcgtgggcc aaggccgcgc cgaactcccg 3093 ctgacccgcg acgagaacgg
atggtgggct cttcagcagc cgtgggacgg cggccccgac 3153 ctcgtcgact
acggctacct cgtcgacggc aagggcccct tcgccgaccc gcggtcgctg 3213
cggcagccgc gcggcgtgca cgagctcggc cgcgaattc 3252 20 26 DNA
Artificial synthetic 20 atgcccgcca gtacctaccg ccttca 26 21 25 DNA
Artificial synthetic 21 tcatgtctcc accagcaggg cgacg 25 22 50 DNA
Artificial synthetic 22 aattcttttt taataaaatc aggaggaatc tagatgttta
ctagtctgca 50 23 42 DNA Artificial synthetic 23 gactagtaaa
catctagatt cctcctgatt ttattaaaaa ag 42 24 33 DNA Artificial
synthetic 24 aaatctagat gcccgccagt acctaccgcc ttc 33 25 33 DNA
Artificial synthetic 25 aaaactagtt tatcatgtct ccaccagcag ggc 33 26
22 DNA Artificial synthetic 26 atcggtgatg tcggcgatat ag 22 27 29
DNA Artificial synthetic 27 gtactggcgg gcatattttt tcctcctga 29 28
31 DNA Artificial synthetic 28 aatcaggagg aaaaaatatg cccgccagta c
31 29 22 DNA Artificial synthetic 29 tcgacgatct gggtgagcgg at 22 30
22 DNA Artificial synthetic 30 tcgacgagca cccggtcgat cc 22 31 26
DNA Artificial synthetic 31 cartgggayg aygaygtnca ycaygc 26 32 2218
DNA Artificial synthetic 32 ctgcagctgc tcgcccccgg aacccccgac
gtgtaccagg gcacggaacg ctgggaccgg 60 tcgctggtgg acccggacaa
ccgtcgcccc gtggatttcg ccgcggcatc cgagctgctc 120 gaccgcctcg
acggcggctg gcggccgccc gtcgacgaga ccggcgcggt caagacgctc 180
gtcgtctccc gcgcgctgcg gctgcgccgc gaccggcccg agctgttcac cgcgtaccac
240 ccggtcacgg cgcgcggcgc gcaggccgag cacctgatcg gcttcgaccg
cggcggcgcg 300 atcgccctgg ccacccgcct gccgctcggc ctcgccgccg
caggcggctg gggcgacacg 360 gtcgtcgacg tcggcgagcg gagcctgcgc
gacgagctga ccggccgcga ggcccgcgga 420 gcggcgcgcg tggccgagtt
gttcgccgac taccccgtcg ccctgctggt ggagac atg 479 Met 1 aac cga cga
ttc ccg gtc tgg gcg ccc cag gcc gcg cag gtg acg ctc 527 Asn Arg Arg
Phe Pro Val Trp Ala Pro Gln Ala Ala Gln Val Thr Leu 5 10 15 gtc gtg
ggc caa ggc cgc gcc gaa ctc ccg ctg acc cgc gac gag aac 575 Val Val
Gly Gln Gly Arg Ala Glu Leu Pro Leu Thr Arg Asp Glu Asn 20 25 30
gga tgg tgg gct ctt cag cag ccg tgg gac ggc ggc ccc gac ctc gtc 623
Gly Trp Trp Ala Leu Gln Gln Pro Trp Asp Gly Gly Pro Asp Leu Val 35
40 45 gac tac ggc tac ctc gtc gac ggc aag ggc ccc ttc gcc gac ccg
cgg 671 Asp Tyr Gly Tyr Leu Val Asp Gly Lys Gly Pro Phe Ala Asp Pro
Arg 50 55 60 65 tcg ctg cgg cag ccg cgc ggc gtg cac gag ctc ggc cgc
gaa ttc gac 719 Ser Leu Arg Gln Pro Arg Gly Val His Glu Leu Gly Arg
Glu Phe Asp 70 75 80 ccc gcc cgc tac gcg tgg ggc gac gac gga tgg
cgc ggc cga gac ctc 767 Pro Ala Arg Tyr Ala Trp Gly Asp Asp Gly Trp
Arg Gly Arg Asp Leu 85 90 95 acc gga gcc gtg atc tac gaa ctg cac
gtc ggc acc ttc acc cct gag 815 Thr Gly Ala Val Ile Tyr Glu Leu His
Val Gly Thr Phe Thr Pro Glu 100 105 110 gga acg ctg gac agc gcc atc
cgt cgc ctc gac cac ctg gtg cgc ctc 863 Gly Thr Leu Asp Ser Ala Ile
Arg Arg Leu Asp His Leu Val Arg Leu 115 120 125 ggc gtc gac gcg gtc
gag ctg ctg ccc gtc aac gcg ttc aac ggc acc 911 Gly Val Asp Ala Val
Glu Leu Leu Pro Val Asn Ala Phe Asn Gly Thr 130 135 140 145 cac ggc
tgg ggc tac gac ggg gtg ctc tgg tac gcg gtg cac gag ccc 959 His Gly
Trp Gly Tyr Asp Gly Val Leu Trp Tyr Ala Val His Glu Pro 150 155 160
tac ggc ggc ccg gag gcg tac cag cgc ttc gtc gac gcc tgc cac gcc
1007 Tyr Gly Gly Pro Glu Ala Tyr Gln Arg Phe Val Asp Ala Cys His
Ala 165 170 175 cgc ggc ctc gcc gtc gtg cag gac gtc gtc tac aac cac
ctg ggc ccg 1055 Arg Gly Leu Ala Val Val Gln Asp Val Val Tyr Asn
His Leu Gly Pro 180 185 190 agc ggc aac cac ctg ccc gac ttc ggc ccc
tac ctc ggg tcg ggc gcc 1103 Ser Gly Asn His Leu Pro Asp Phe Gly
Pro Tyr Leu Gly Ser Gly Ala 195 200 205 gcc aac acc tgg ggc gac gcg
ctg aac ctc gac ggg ccg ctc tcc gac 1151 Ala Asn Thr Trp Gly Asp
Ala Leu Asn Leu Asp Gly Pro Leu Ser Asp 210 215 220 225 gag gtg cgg
cgg tac atc atc gac aac gcg gtg tac tgg ctg cgc gac 1199 Glu Val
Arg Arg Tyr Ile Ile Asp Asn Ala Val Tyr Trp Leu Arg Asp 230 235 240
atg cac gcc gac ggg ctg cgg ctc gac gcc gtg cac gcg ctg cgc gac
1247 Met His Ala Asp Gly Leu Arg Leu Asp Ala Val His Ala Leu Arg
Asp 245 250 255 gcc cgc gcg ctg cac ctg ctc gaa gag ctc gcc gcc cgc
gtc gac gag 1295 Ala Arg Ala Leu His Leu Leu Glu Glu Leu Ala Ala
Arg Val Asp Glu 260 265 270 ctg gcg ggc gag ctc ggc cgg ccg ctg acg
ctc atc gcc gag agc gac 1343 Leu Ala Gly Glu Leu Gly Arg Pro Leu
Thr Leu Ile Ala Glu Ser Asp 275 280 285 ctg aac gac ccg aag ctg atc
cgc tcc cgc gcg gcg cac ggc tac ggc 1391 Leu Asn Asp Pro Lys Leu
Ile Arg Ser Arg Ala Ala His Gly Tyr Gly 290 295 300 305 ctc gac gcc
cag tgg gac gac gac gtg cac cac gcg gtg cac gcc aac 1439 Leu Asp
Ala Gln Trp Asp Asp Asp Val His His Ala Val His Ala Asn 310 315 320
gtg acc ggc gag acc gtc ggc tac tac gcc gac ttc ggc ggg ctc ggc
1487 Val Thr Gly Glu Thr Val Gly Tyr Tyr Ala Asp Phe Gly Gly Leu
Gly 325 330 335 gcc ctc gtc aag gtg ttc cag cgc ggc tgg ttc cac gac
ggc acc tgg 1535 Ala Leu Val Lys Val Phe Gln Arg Gly Trp Phe His
Asp Gly Thr Trp 340 345 350 tcg agc ttc cgc gag cgg cac cac ggc cgg
ccg ctc gac ccc gac atc 1583 Ser Ser Phe Arg Glu Arg His His Gly
Arg Pro Leu Asp Pro Asp Ile 355 360 365 ccg ttc cgc cgg ctc gtc gcc
ttc gcg cag gat cac gac cag gtc ggc 1631 Pro Phe Arg Arg Leu Val
Ala Phe Ala Gln Asp His Asp Gln Val Gly 370 375 380 385 aac cga gcg
gtc ggc gac cgc atg tcg gcg cag gtc ggc gag ggt tcg 1679 Asn Arg
Ala Val Gly Asp Arg Met Ser Ala Gln Val Gly Glu Gly Ser 390 395 400
ctc gcc gcc gcg gcg gcg ctc gtg ctg ctc ggc ccg ttc acc ccg atg
1727 Leu Ala Ala Ala Ala Ala Leu Val Leu Leu Gly Pro Phe Thr Pro
Met 405 410 415 ctg ttc atg ggc gag gag tgg ggc gcg cgc acc ccg tgg
cag ttc ttc 1775 Leu Phe Met Gly Glu Glu Trp Gly Ala Arg Thr Pro
Trp Gln Phe Phe 420 425 430 acc tcc cac ccc gag ccc gag ctg ggg gag
gcg acg gcg cgc ggg cgc 1823 Thr Ser His Pro Glu Pro Glu Leu Gly
Glu Ala Thr Ala Arg Gly Arg 435 440 445 atc gcc gag ttc gcc cgc atg
ggc tgg gac ccg gca gtc gtg ccc gac 1871 Ile Ala Glu Phe Ala Arg
Met Gly Trp Asp Pro Ala Val Val Pro Asp 450 455 460 465 ccg cag gac
ccg gcc acc ttc gcc cgc tcg cac ctg gac tgg tcc gag 1919 Pro Gln
Asp Pro Ala Thr Phe Ala Arg Ser His Leu Asp Trp Ser Glu 470 475 480
ccc gag cgg gaa ccg cac gcg ggc ctg ctc gcc ttc tac acc gac ctg
1967 Pro Glu Arg Glu Pro His Ala Gly Leu Leu Ala Phe Tyr Thr Asp
Leu 485 490 495 atc gcg ctg cgg cgc gag ctg ccg gtc gat gcg ccg gcg
cgc gag gtg 2015 Ile Ala Leu Arg Arg Glu Leu Pro Val Asp Ala Pro
Ala Arg Glu Val 500 505 510 gat gcc gac gag gcg cgc ggc gtc ttc gcg
ttc agc cgc ggc ccg ctg 2063 Asp Ala Asp Glu Ala Arg Gly Val Phe
Ala Phe Ser Arg Gly Pro Leu 515 520 525 cgg gtc acg gtc gcg ctg cgc
ccc gga ccg gtc ggg gtg ccc gag cac 2111 Arg Val Thr Val Ala Leu
Arg Pro Gly Pro Val Gly Val Pro Glu His 530 535 540 545 ggg ggc ctc
gtg ctc gcc tac ggc gag gtg cgc gcc ggc gcc gcc gga 2159 Gly Gly
Leu Val Leu Ala Tyr Gly Glu Val Arg Ala Gly Ala Ala Gly 550 555 560
ctg cac ctc gac ggg ccg gga gcc gcg atc gtg cgc ctc gag 2201 Leu
His Leu Asp Gly Pro Gly Ala Ala Ile Val Arg Leu Glu 565 570 575
tgacgcggct gggtacc 2218 33 25 DNA Artificial synthetic 33
atgaaccgac gattcccggt ctggg 25 34 25 DNA Artificial synthetic 34
tcactcgagg cgcacgatcg cggct 25 35 36 DNA Artificial synthetic 35
aaatctagat gaaccgacga ttcccggtct gggcgc
36 36 36 DNA Artificial synthetic 36 aaaactagtt tatcactcga
ggcgcacgat cgcggc 36 37 28 DNA Artificial synthetic 37 atcgtcggtt
catatttttt cctcctga 28 38 28 DNA Artificial synthetic 38 aatcaggagg
aaaaaatatg aaccgacg 28 39 22 DNA Artificial synthetic 39 aggtggttgt
agacgacgtc ct 22
* * * * *